Linear and circular expression elements

ABSTRACT

The present invention relates to linear expression elements (LEEs) and circular expression elements (CEEs), which are useful in a variety of molecular biology protocols. Specifically, the invention relates to the use of LEEs and CEEs to screen for gene function, biological effects of gene function, antigens, and promoter function. The invention also provides methods of producing proteins, antibodies, antigens, and vaccines. Also, the invention relates to methods of making LEEs and CEEs, and LEEs and CEEs produced by such methods.

[0001] This application claims the priority of U.S. ProvisionalApplication Ser. No. 60/125,864, filed Mar. 24, 1999 and U.S.Provisional Application Ser. No. 60/127,222, filed Mar. 31, 1999, eachof which disclosures is specifically incorporated herein by reference inits entirety.

[0002] The government owns rights in the present invention pursuant toDARPA Federal grant number BAA 96-24.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates generally to the field of molecularbiology and functional genomics. More particularly, it concerns methodsto screen genes or gene components for their functions or biologicaleffects and methods of generating immune responses or immune reagents.

[0005] 2. Description of Related Art

[0006] Genomic sequencing efforts are producing a wealth of data.Sequence information is being compiled not only from humans but alsoplants, animals and microbes. This abundance of data has spawned theneed for new technologies for analyzing and functionally assessing themillions of genes that will now be available. The expanding repertoireof areas that functional genomics are applied should lead to newinsights into evolution, reveal how cellular pathways integrate, andyield new drugs and vaccines. A current challenge is to develop thetechnologies that will enable these advancements.

[0007] For example, there are currently at least thirty microbial genomesequences in the public domain and additional projects underway. Most ofthese are pathogens of humans or commercial animals. One often expressedhope is that knowledge of these sequences will lead to the developmentof vaccines against these pathogens. Though computational analyses maybe useful, the more sure-footed approach would be to functionally screeneach gene from all the pathogens in animals for its value as aprotective agent. However the time and expense of cloning thousands ofgenes from each pathogen then preparing appropriate reagents from themis prohibitive, using current methods.

[0008] In order to quickly and effectively assess the activity of anyparticular gene product or a physiological response to it, an assaymethod is required that avoids plasmids and bacterial cloningprocedures. This ideal method would also permit the plethora of newgenes from sequencing projects to be rapid screened in organisms, cellsor cell-free systems.

SUMMARY OF THE INVENTION

[0009] The inventors have determined methods and compositions whichallow for the production of linear expression elements (“LEEs”) and/orcircular expression elements (“CEEs”) encompassing a complete gene(promoter, coding sequence, and terminator). These LEEs and CEEs can bedirectly introduced into and expressed in cells or an intact organism toyield expression levels comparable to those from a standard supercoiled,replicative plasmid.

[0010] In some general embodiments, the invention relates to methods ofassaying for the production or regulation of expression of at least onepolypeptide from a linear or circular nucleic acid segment comprising apromoter or putative promoter and an ORF or putative ORF. These methodsmay comprise: a) obtaining at least one linear or circular nucleic acidsegment comprising a promoter or putative promoter and an ORF encoding apeptide or putative ORF; b) placing the nucleic acid segment underconditions conducive to expression of the polypeptide from the ORF; andc) assaying for the production or regulation of expression of apolypeptide from the ORF or putative ORF. In many preferred embodiments,the nucleic acid segment will comprise a terminator. However, in someapplications, including those where the linear nucleic acid segment isassayed in a cell-free expression system, no terminator is required. Insome cases, the linear nucleic acid is obtained by a PCR® process. Inother situations, the linear nucleic acid may be cut out of a plasmid,chromosome, or other larger piece of nucleic acid; in these cases, thelinear nucleic acid cut from the larger piece of nucleic acid willtypically comprise a promoter and ORF, and in many cases a terminator,that are already in operable relationship.

[0011] Of course, those of skill in the art will understand that thepromoter or putative promoter and any terminator will typically beplaced in a operable relationship to the ORF or putative ORF, employingmethods disclosed herein or know to those in the art.

[0012] In many cases, the linear nucleic acid segment is obtained bysynthesis. In some preferred methods, the synthesis comprisesnoncovalent linkage of the promoter to the ORF. For example, thisnon-covalent linkage may be performed by a) obtaining a PCR® productcomprising the nucleic acid segment, which PCR® product is obtained byamplification from at least one primer that has complementary stretchescomprising deoxyuridines with uracil-DNA glycosylase to create overhangsto which the promoter can link; b) providing the promoter; and c)non-covalently linking the promoter to the nucleic acid segment tocreate the linear or circular expression element. In many cases aterminator will be non-covalently linked to the ORF, using a similartechnique, although it is possible that the ORF or putative ORF may havea terminator incorporated into it, such that the addition of aterminator may not be required. In some presently employed embodiments,the primer that has complementary stretches comprising deoxyuridines.These stretches allow the use of uracil-DNA glycosylase, or anothersuitable enzyme to create overhangs to which the promoter cannon-covalently link create the linear or circular expression element.The non-covalent linkage of a terminator to the ORF can be accomplishedby much the same technique. Of course, it will be understood to those ofskill in the art that other nucleotide/enzyme pairs may be used toperform this non-covalent linking, and that other techniques ofnon-covalent linking may be employed, so long as the purposes of theinvention are accomplished. In some specific embodiments, the primercomprises the promoter and the terminator in divergent orientation, suchthat the step of non-covalently linking the promoter and the terminatorto the ORF results in a circular expression element.

[0013] While the promoter may be of any origin that will work for thepurposes of the invention, in some preferred embodiments, the promoteris a eukaryotic promoter. Likewise, the terminator may be of any source,but in may cases the terminator will be a eukaryotic terminator.

[0014] he nucleic acid segment containing the ORF, putative ORF, or anyother nucleic acid segment which is comprised in a LEE or CEE may beobtained from any of a variety of sources. For example, it may beobtained by PCR®, from a linear nucleic acid that is cut out of aplasmid, or obtained by synthesis.

[0015] In some methods according to the invention, the nucleic acidsegment forming LEE or CEE is placed into a cell so that it is underconditions conducive to expression of a polypeptide from the ORF orputative ORF. In some preferred embodiments, the linear nucleic acid isplaced into a cell but not integrated into the cell's genome. Theinventors have determined that integration into the genome is notrequired for expression of linear nucleic acids. Further, the inventorshave determined that supercoiled plasmids are not required forexpression of genes. In some embodiments, the cell is in cell culture,while in others, the cell is comprised in a tissue or an entireorganism. All organisms are contemplated in this regard, including, butnot limited to plant, animal, mammal, fish, bird, reptilian, human,rabbit, rat, hamster, mouse and other cells. The LEEs and CEEs of theinvention may be placed into cells using any of the technologiesdescribed elsewhere in the specification. In some preferred embodiments,a LEE or CEE in injected into the cell. In some particularly preferredembodiments injection comprises microprojectile bombardment. In otherembodiments, the LEE or CEE many be placed in a cell-free expressionreaction.

[0016] Various preferred embodiments relate to methods of analyzing anucleic acid sequence comprising: a) obtaining a nucleic acid segment;b) linking the nucleic acid segment to a promoter and a terminator tocreate a linear or circular expression element; c) providing the linearor circular expression element to a cell-free expression system or to acell under conditions conducive to expression of any product encoded forby the nucleic acid segment; and d) analyzing any expression of anyproduct encoded by the nucleic acid sequence. The nucleic acid segmentsemployed in these methods can be obtained in manners described above,and elsewhere in the specification. Likewise the linkage of the promoterand terminator may be non-covalent linkage, as described elsewhere. Insome embodiments wherein the nucleic acid sequence comprises an ORF tobe analyzed for function, for example, to determine whether the nucleicacid encodes a polypeptide. These methods also allow one to determinewhether or not the ORF encodes an antigenic polypeptide, and/or todetermine biological properties of the polypeptide. For example one candetermine whether the linear or circular expression element, or an ORFcontained therein, is suitable for use in a vaccine. An advantage ofthese methods is that more than one distinct nucleic acid segment isanalyzed in a single procedure.

[0017] As discussed above, and elsewhere in this specification, themethods of the invention may involve assaying for expression of apolypeptide which may be encoded in an ORF or putative ORF. In somepreferred embodiments, the methods comprise assaying for expression ofthe polypeptide; in some cases, such assaying includes identification ofthe polypeptide. Other embodiments may comprise assaying the expressionof a reporter gene product encoded in the ORF.

[0018] Still other specific embodiments comprise assaying for functionof the promoter. For example, the function of the promoter may beassayed by determining whether a reporter gene product encoded in theORF is expressed. A reporter gene is a gene that confers on itsrecombinant hosts a readily detectable phenotype that emerges only underconditions where a general DNA promoter positioned upstream of thereporter gene is functional. Generally, reporter genes encode apolypeptide (marker protein) not otherwise produced by the host cellwhich is detectable by analysis of the cell culture, e.g., byfluorometric, radioisotopic or spectrophotometric analysis of the cellculture. In some embodiments, the assessment of promoter function willcomprise comparing the function of two or more putative promoters forwhich function is assayed. In this manner, the invention provides for anefficient manner of screening a variety of promoters for function in aspecific system. For example, a library of promoters from a variety ofsources can be assayed such that an optimal promoter for a particularsystem is determined. Alternatively, a variety of mutants of a specificpromoter may be assayed to determine whether they have promoteractivity. In order to perform such assays, one may construct a varietyof linear nucleic acid segments, using standard molecular biology means,each of which comprises a putative promoter or a promoter and apolypeptide encoding a reporter gene. This variety of linear nucleicacids may then be introduced to a system that enables the assessment oftheir function by looking for expression of the reporter gene.

[0019] In addition to allowing for analysis of ORFs or putative ORFs,the invention provides methods of analyzing a nucleic acid segment foractivity as a promoter comprising: a) obtaining a nucleic acid segmentencoding a putative promoter; b) linking the nucleic acid segmentencoding the putative promoter to a nucleic acid encoding a polypeptideto create a linear or circular expression element; c) providing thelinear or circular expression element to a cell-free expression systemor to a cell under conditions conducive to expression of thepolypeptide; and d) analyzing any expression of the polypeptide. Forexample, the function of the promoter may be assayed by determiningwhether a reporter gene product encoded in the ORF is expressed. Areporter gene is a gene that confers on its recombinant hosts a readilydetectable phenotype that emerges only under conditions where a generalDNA promoter positioned upstream of the reporter gene is functional.Generally, reporter genes encode a polypeptide (marker protein) nototherwise produced by the host cell which is detectable by analysis ofthe cell culture, e.g., by fluorometric, radioisotopic orspectrophotometric analysis of the cell culture. In some embodiments,the assessment of promoter function will comprise comparing the functionof two or more putative promoters for which function is assayed. In thismanner, the invention provides for an efficient manner of screening avariety of promoters for function in a specific system. For example, alibrary of promoters from a variety of sources can be assayed such thatan optimal promoter for a particular system is determined.Alternatively, a variety of mutants of a specific promoter may beassayed to determine whether they have promoter activity. In order toperform such assays, one may construct a variety of linear nucleic acidsegments, using standard molecular biology means, each of whichcomprises a putative promoter or a promoter and a polypeptide encoding areporter gene. This variety of linear nucleic acids may then beintroduced to a system that enables the assessment of their function bylooking for expression of the reporter gene. In this manner, one is ableto assay for function of the promoter or putative promoter, anddetermine whether the promoter or putative promoter is functioning, andthe extent of any such function, by determining whether the reportergene product encoded in an ORF is expressed. In some embodiments, themethods may be used to compare the function of two or more putativepromoters. It is advantageous that more than one distinct nucleic acidsegment encoding a putative promoter can analyzed in a single procedure.The nucleic acid encoding the putative promoter may be, for example, anative nucleic acid, prepared by mutation of a native promoter sequence,or prepared by chemical synthesis. One of skill can use this thesetechniques to test and optimize promoters or putative promoters from anysource.

[0020] In additional embodiments, the invention relates to methods ofscreening for a biological response comprising: a) obtaining a linear orcircular expression element by a process comprising: obtaining a DNAsegment comprising an ORF; linking the ORF to a promoter and aterminator to create a linear or circular expression element; andb)providing the expression element to an organism under conditionsconducive to expression of any product encoded for by the ORF. In suchcases, more than one type of linear or circular expression element isintroduced to the organism. These methods may encompass method ofproducing antibodies for analytical purposes, for example, when thebiological function is to result in an immune response. In otherembodiments, these methods may provide methods of vaccinating theorganism, as described below.

[0021] In some specific embodiments, the invention allows for thevaccinating an organism comprising obtaining a linear or circularexpression element by a process comprising obtaining a DNA segmentcomprising an ORF and linking the ORF to a promoter and a terminator tocreate a linear or circular expression element; and c) providing theexpression element to an organism under conditions conducive toexpression of any product encoded for by the ORF, such that immuneresponse is produced in the animal. In such vaccines, more than one typeof linear or circular expression element is introduced to the organism,in order to create a polyvalent vaccine that my be directed against morethat one infectious agent, cancer, or other disease or against a varietyof antigens from a single infectious agent, cancer, or other disease.For example, a plurality of types of linear or circular expressionelements may introduced to the organism., and the plurality of types oflinear or circular expression elements may comprise elements encodingdistinct polypeptides of a pathogen. While those of skill in the artwill realize that the pathogen may be of any form, in some preferredembodiments, the pathogen is a virus, bacterium, fungus, alga,protozoan, arthropod, nematode, platyhelninthe, or plant. In even morespecific embodiments, the individual linear or circular expressionelements encoding all potential allergens of a virus is comprised in theplurality of types of linear or circular expression elements. The ORFmay also encodes a polypeptide which is useful for vaccination againstcancer, or another disease, as known to those of skill in the art.

[0022] The invention also contemplates methods of selecting ORFseffective for generating an immune response specific to a pathogen,cancer, or other disease in an organism, comprising: a) preparing aplurality of linear or circular expression elements comprising aplurality of DNA segments comprising ORFs from a pathogen, cancer, orother disease; b) introducing the plurality of linear or circularexpression elements into an organism; and c) selecting from theplurality of linear or circular expression elements ORFs that areeffective to generate said immune response. Such methods may furthercomprising testing said organism against challenge with the pathogenwherein the organism is protected against challenge with the pathogen.In this manner, one or more antigens conferring a protective responsemay identified by screening of the organism.

[0023] The invention also relates to linear and circular expressionelement and method of producing linear and circular expression elements.For example, such methods may comprise a) obtaining a DNA segmentcomprising an ORF, putative ORF, or other sequence; and b) linking theDNA segment to a promoter and a terminator to create a linear orcircular expression element. In some cases, the DNA segment is obtainedfrom a process involving PCR®, and in some specific embodiments, thePCR® reaction is primed with oligonucleotides having a complementarystretch incorporating deoxyuridines. For example, the deoxyuridines maybe incorporated every third position of the complementary stretch. TheORF may be non-covalently linked to the promoter and the terminator, andthis non-covalent linkage may be performed as described elsewhere inthis specification.

[0024] In some specific embodiments, the invention relates to linear orcircular expression elements comprising a DNA segment comprising an ORFand a promoter and terminator non-covalently linked to said ORF. Inother aspects, the invention relates to antibodies, antigens andvaccines that are prepared, assayed, or determined employing theabove-described methods.

[0025] As used herein the specificaition, “a” or “an” may mean one ormore. As used herein in the claim(s), when used in conduction with theword comprising, the words “a” or “an” may mean one or more than one.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The following drawings form part of the present specification andare included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

[0027]FIG. 1. Delivery and expression of a PCR®-amplified luciferasegene in mice. Ears were bombarded with empty plasmid, pCMVi (0.33 μg),luciferase plasmid, pCMViLUC (0.33 μg), or a molar-equivalent dose (0.17μg) of a PCR® product encoding luciferase. c. LEE.LUC=crude productdirectly loaded onto microprojectiles; f.p.LEE.LUC=filter purifiedproduct; g.p.LEE.LUC agarose gel purified product; noTaq=filter-purified sample prepared without Taq polymerase. Activitiesare plotted as lumens×10⁶ per 1.0 μg plasmid or per 0.5 μg PCR® productintroduced into an ear. Lumens were measured from four or more bombardedears then averaged. Standard errors are defined.

[0028]FIG. 2. Cartoon depicting a method for building LEEs from separategenetic components.

[0029]FIG. 3. The noncovalently linked promoter and ORF produce reporteractivity. Ears were bombarded with the indicated DNAs, someabbreviations are described in FIG. 1. No UDG=UDG-sensitive productsdelivered without UDG pre-treatment; no dU=promoter and LUC productsamplified with standard primers; CMVi=only promoter product; LUC=onlyreporter product (topologies of the introduced expression elements aredrawn at the bottom of FIG. 3); Pr=promoter; ORF=open reading frame,X=no product. Activities are reported as described in FIG. 1.

[0030]FIG. 4. A trimolecular LEE efficiently produces luciferase geneactivity in vivo. bi-LEE.LUC(−T)=two linked PCR® products carryingpromoter and LUC without a terminator; tri-LEE.LUC=three linked PCR®products carrying promoter, ORF, and terminator. Some abbreviations aredescribed in FIG. 3. Topologies of the introduced expression elementsare drawn at the bottom of FIG. 4. ORF(−T)=LUC ORF without terminator;T=terminator. Activities are reported as described in FIG. 1.

[0031]FIG. 5. Introduction of a bimolecular LEE encoding AAT generatesspecific antibodies in mice at titers comparable to that produced from aplasmid encoding this test antigen. Mice were biolistically immunizedwith either 1 μg of pCMVi, 1 μg pCMViAAT, or 0.5 μg bi-LEE.AAT. Serawere collected and tested by ELISA at 1:1000 dilution. Topologies of theintroduced DNAs are drawn below the graph. Individual and group averagesare plotted with standard errors.

[0032]FIG. 6. LEE-encoded compared with plasmid-encoded gene expressionin tissue culture. Plasmid pCMVi is the empty plasmid, pCMViLUC is thestandard plasmid encoding luciferase (LUC), and the LEE.LUC DNAs are thelinear expression elements.

[0033]FIG. 7. LEEs can be delivered into animals biolistically, byneedle injection and by lipid-mediated uptake.

[0034]FIG. 7A Gene-gun delivery of bi-LEE.LUC leads to similar geneexpression with or without covalent linkage.

[0035]FIG. 7B Needle delivery of the LEE can be improved by in vitroligation. Note 100-fold difference in x-axis scale between ear (×10⁶)and muscle (×10⁴) sample readouts. Activities are reported as in FIG. 1.−lig=annealed but not ligated in vitro. +lig=treated with ligase 30 minbefore in vivo introduction.

[0036]FIG. 7C. Liposome delivery of bi-LEE.LUC into WEH1 tissue culturecells leads to higher levels of luciferase activity than delivery of theLUC-encoding plasmid pCMV.LUC. The experiment was performed three timeswith lumen determinators measured in duplicate 18 hours aftertrasnfection (Superfect, Qiagen).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0037] The increasing availability of genomic sequence information hasaccentuated the need for new methods to efficiently assess gene functionand prepare reagents to study these functions. The disclosed methods andcompositions of the invention allow any open-reading frame (ORF), forexample, PCR® amplified ORFs, to be non-convalently linked to aneukaryotic promoter and terminator. These quickly linked fragments canbe directly injected into animals to produce local gene expression. Ithas also been demonstrated that the ORFs can be injected into mice toproduce antibodies to the encoded foreign protein by simply attachingmammalian promoter and terminator sequences. This technology makes itpossible to rapidly screen large numbers of genes for their function invivo or produce an immune response to them without the necessity ofcloning, bacterial propagation or protein purification.

[0038] Preparation of genes for transfection and expression has becomesynonymous with the cloning, then amplification and purification ofplasmid constructs from bacteria. The process involves many steps thatconsume time, money, and carries the bias, toxicity, and contaminationrisks inherent to bacterial propagation. The inventors' have developed atwo-pronged strategy that streamlines these activities. In someembodiments, it is based on: 1) delivery of genetic expression unitsinto organisms without the need of bacterial propagation and 2)cassette-like linkage of promoters, genes, and terminators. Efficientuse of PCR® products, restriction fragments or chemically synthesizedgenes as vehicles of expression enables genetic constructs to begenerated on a scale, purity, and time-frame that are not possible byusual cloning methods. This protocol has several other advantages. Sincegenes are produced without bacteria, problems associated with bacterialgrowth such as toxicity, lethality, or stability are avoided. Incontrast to conventional cloning where an individual PCR® product isisolated in a plasmid and propagated, this method introduces the entireamplified sample thus avoiding concern that any particular PCR® productcarries a mutation. This single-tube, bio-free protocol would beadaptable to robotic handling and high-through-put screens. Finally, thesynthetic genes will maintain the advantage of unmethylated purines(CpGs) as adjuvant in raising immune responses (Sato et al., 1996).

[0039] This protocol will be useful in several manners. First, a genomedatabase will allow oligos to be designed to amplify each of the genesof an organism. As shown in the examples below, dU residues and UDGenzymes were used to generate attachable sequences. While this was veryeffective, relatively high levels of reporter gene expression were alsoobserved even without enzyme treatment. It may be that exposure toendogenous UDG enables in vivo annealing and sticky-end ligation. LEEsbuilt from either two or three PCR® products generated similar levels ofgene activity.

[0040] Each ORF can be generated, annealed to a promoter and terminatorof choice, then directly introduced into a test cell, tissue or organimsor used in a cell-free system. LEEs and CEEs can be screenedindividually or in pools. With this method, it is envisioned, forexample, that all the genes of an organism can be introduced as geneticvaccines into organisms (e.g., an animal) in a matter of days. Theanimals can be subsequently challenged with pathogen to determine whichgenes protect against disease. Isolating individual LEEs or CEEs fromprotective pools can be conducted as previously described for plasmidlibraries (Barry et al., 1995).

[0041] A second application is in developing immunological reagents.LEEs and CEEs can be used to produce antibodies to an ORF. Antibodies toall a pathogen's ORFs can be produced, then used to probepathogen-infected tissue in immunolocalization analyses to elucidatewhich proteins are present at any pathogen stage-of-interest. Identifiedgene products are excellent vaccine candidates or drug targets.Alternatively, the antibodies could be screened for diagnostic ortherapeutic value.

[0042] A third application is to screen for other physiologicalresponses caused by expression of a gene product.

[0043] A fourth application of LEEs and CEEs is to screen for alteredpromoter function. Libraries of promoters containing deletions ormutations are linked to a reporter gene and introduced directly into therelevant animal/plant tissue or cell culture. For example, reporteractivity can be used to monitor gene expression levels, cell-typespecific expression, or responses to treatments such as drugs. Sincethis protocol allows an efficient and fast method to generate expressionelements of any design for direct introduction into tissue, it isapplicable to other applications.

[0044] A fifth application of LEEs and CEEs could be in cell-freeexpression systems. In vitro transcribed/translated LEEs and CEEs couldbe used to rapidly and systematically generate proteins encoded by anynumber of ORFs. The proteins could be screened for a function oractivity of interest, for example, the ability to bind a drug or otherprotein target.

[0045] A. Promoters

[0046] In certain aspects of the present invention, a LEE or CEE that isemployed will comprise at least one promoter. A “promoter” is a controlsequence that is a region of a nucleic acid sequence at which initiationand rate of transcription are controlled. It may contain geneticelements at which regulatory proteins and molecules may bind such as RNApolymerase and other transcription factors. The phrases “operativelypositioned,” “operatively linked,” “under control,” and “undertranscriptional control” mean that a promoter is in a correct functionallocation and/or orientation in relation to a nucleic acid sequence(i.e., ORF)to control transcriptional initiation and/or expression ofthat sequence. A promoter may or may not be used in conjunction with an“enhancer,” which refers to a cis-acting regulatory sequence involved inthe transcriptional activation of a nucleic acid sequence.

[0047] Certain advantages will be gained by positioning the codingnucleic acid segment under the control of a recombinant or heterologouspromoter, which refers to a promoter that is not normally associatedwith a nucleic acid sequence in its natural environment. A recombinantor heterologous enhancer refers also to an enhancer not normallyassociated with a nucleic acid sequence in its natural environment. Suchpromoters or enhancers may include promoters or enhancers of othergenes, and promoters or enhancers isolated from any other prokaryotic,viral, or eukaryotic cell, and promoters or enhancers not “naturallyoccurring,” i.e., containing different elements of differenttranscriptional regulatory regions, and/or mutations that alterexpression. In addition to producing nucleic acid sequences of promotersand enhancers synthetically, sequences may be produced using recombinantcloning and/or nucleic acid amplification technology, including PCR™, inconnection with the compositions disclosed herein (see U.S. Pat. Nos.4,683,202, 5,928,906, each incorporated herein by reference).Furthermore, it is contemplated the control sequences that directtranscription and/or expression of sequences within non-nuclearorganelles such as mitochondria, chloroplasts, and the like, can beemployed as well. However, in certain embodiments a promoter may be onenaturally associated with a gene or sequence, as may be obtained byisolating the 5′ non-coding sequences located upstream of the codingsegment and/or exon. Such a promoter can be referred to as “endogenous.”Similarly, an enhancer may be one naturally associated with a nucleicacid sequence, located either downstream or upstream of that sequence.

[0048] Naturally, it will be important to employ a promoter and/orenhancer that effectively directs the expression of the DNA segment inthe organelle, cell, tissue and organism chosen for expression. Those ofskill in the art of molecular biology generally know the use ofpromoters, enhancers, and cell type combinations for protein expression,for example, see Sambrook et al. (1989), incorporated herein byreference. The promoters employed may be constitutive, tissue-specific,inducible, and/or useful under the appropriate conditions to direct highlevel expression of the introduced DNA segment, such as is advantageousin the large-scale production of recombinant proteins and/or peptides.The promoter may be heterologous or endogenous.

[0049] Tables 1 lists several elements/promoters that may be employed,in the context of the present invention, to regulate the expression of agene. This list is not intended to be exhaustive of all the possibleelements involved in the promotion of expression but, merely, to beexemplary thereof. Table 2 provides non-limiting examples of inducibleelements, which are regions of a nucleic acid sequence that can beactivated in response to a specific stimulus. TABLE 1 Promoter and/orEnhancer Promoter/Enhancer References Immunoglobulin Heavy Banerji etal., 1983; Gilles et al., 1983; Chain Grosschedl et al., 1985; Atchinsonet al., 1986, 1987; Imler et al., 1987; Weinberger et al., 1984;Kiledjian et al., 1988; Porton et al.; 1990 Immunoglobulin Light Queenet al., 1983; Picard et al., 1984 Chain T-Cell Receptor Luria et al.,1987; Winoto et al., 1989; Redondo et al.; 1990 HLA DQ a and/or DQ βSullivan et al., 1987 β-Interferon Goodbourn et al., 1986; Fujita etal., 1987; Goodbourn et al., 1988 Interleukin-2 Greene et al., 1989Interleukin-2 Receptor Greene et al., 1989; Lin et al., 1990 MHC ClassII 5 Koch et al., 1989 MHC Class II HLA-Dra Sherman et al., 1989 β-ActinKawamoto et al., 1988; Ng et al.; 1989 Muscle Creatine Kinase Jaynes etal., 1988; Horlick et al., 1989; (MCK) Johnson et al., 1989 PrealbuminCosta et al., 1988 (Transthyretin) Elastase I Omitz et al., 1987Metallothionein (MTII) Karin et al., 1987; Culotta et al., 1989Collagenase Pinkert et al., 1987; Angel et al., 1987 Albumin Pinkert etal., 1987; Tronche et al., 1989, 1990 α-Fetoprotein Godbout et al.,1988; Campere et al., 1989 T-Globin Bodine et al., 1987; Perez-Stable etal., 1990 β-Globin Trudel et al., 1987 C-fos Cohen et al., 1987 C-HA-rasTriesman, 1986; Deschamps et al., 1985 Insulin Edlund et al., 1985Neural Cell Adhesion Hirsh et al., 1990 Molecule (NCAM) α₁-AntitrypainLatimer et al., 1990 H2B (TH2B) Histone Hwang et al., 1990 Mouse and/orType I Ripe et al., 1989 Collagen Glucose-Regulated Chang et al., 1989Proteins (GRP94 and GRP78) Rat Growth Hormone Larsen et al., 1986 HumanSerum Amyloid Edbrooke et al., 1989 A (SAA) Troponin I (TN I) Yutzey etal., 1989 Platelet-Derived Growth Pech et al., 1989 Factor (PDGF)Duchenne Muscular Klamut et al., 1990 Dystrophy SV40 Banerji et al.,1981; Moreau et al., 1981; Sleigh et al., 1985; Firak et al., 1986; Herret al., 1986; Imbra et al., 1986; Kadesch et al., 1986; Wang et al.,1986; Ondek et al., 1987; Kuhl et al., 1987; Schaffner et al., 1988Polyoma Swartzendruber et al., 1975; Vasseur et al., 1980; Katinka etal., 1980, 1981; Tyndell et al., 1981; Dandolo et al., 1983; de Villierset al., 1984; Hen et al., 1986; Satake et al., 1988; Campbell and/orVillarreal, 1988 Retroviruses Kriegler et al., 1982, 1983; Levinson etal., 1982; Kriegler et al., 1983, 1984a, b, 1988; Bosze et al., 1986;Miksicek et al., 1986; Celander et al., 1987; Thiesen et al., 1988;Celander et al., 1988; Chol et al., 1988; Reisman et al., 1989 PapillomaVirus Campo et al., 1983; Lusky et al., 1983; Spandidos and/or Wilkie,1983; Spalholz et al., 1985; Lusky et al., 1986; Cripe et al., 1987;Gloss et al., 1987; Hirochika et al., 1987; Stephens et al., 1987; Glueet al., 1988 Hepatitis B Virus Bulla et al., 1986; Jameel et al., 1986;Shaul et al., 1987; Spandau et al., 1988; Vannice et al., 1988 HumanImmunodefi- Muesing et al., 1987; Hauber et al., 1988; ciency VirusJakobovits et al., 1988; Feng et al., 1988; Takebe et al., 1988; Rosenet al., 1988; Berkhout et al., 1989; Laspia et al., 1989; Sharp et al.,1989; Braddock et al., 1989 Cytomegalovirus (CMV) Weber et al., 1984;Boshart et al., 1985; Foecking et al., 1986 Gibbon Ape Leukemia Holbrooket al., 1987; Quinn et al., 1989 Virus

[0050] TABLE 2 Inducible Elements Element Inducer References MT IIPhorbol Ester (TFA) Palmiter et al., 1982; Heavy metals Haslinger etal., 1985; Searle et al., 1985; Stuart et al., 1985; Imagawa et al.,1987, Karin et al., 1987; Angel et al., 1987b; MeNeall et al., 1989 MMTV(mouse Glucocorticoids Huang et al., 1981; mammary tumor Lee et al.,1981; virus) Majors et al., 1983; Chandler et al., 1983; Lee et al.,1984; Ponta et al., 1985; Sakai et al., 1988 β-Interferon poly(rI)xTavernier et al., 1983 poly(rc) Adenovirus 5 E2 ElA Imperiale et al.,1984 Collagenase Phorbol Ester (TPA) Angel et al., 1987a StromelysinPhorbol Ester (TPA) Angel et al., 1987b SV40 Phorbol Ester (TPA) Angelet al., 1987b Murine MX Gene Interferon, Newcastle Hug et al., 1988Disease Virus GRP78 Gene A23187 Resendez et al., 1988 α-2-MacroglobulinIL-6 Kunz et al., 1989 Vimentin Serum Rittling et al., 1989 MHC Class IGene Interferon Blanar et al., 1989 H-2κb HSP70 ElA, SV40 Large T Tayloret al., 1989, 1990a, Antigen 1990b Proliferin Phorbol Ester-TPA Mordacqet al., 1989 Tumor Necrosis PMA Hensel et al., 1989 Factor ThyroidStimulating Thyroid Hormone Chatterjee et al., 1989 Hormone α Gene

[0051] In certain embodiments, the promoter may be a elongation factor1a (EF1a), an inducible promoter such as tet^(R), the one of the RU486system or the meristerone system.

[0052] The identity of tissue-specific promoters or elements, as well asassays to characterize their activity, is well known to those of skillin the art. Examples of such regions include the human LIMK2 gene(Nomoto et al. 1999), the somatostatin receptor 2 gene (Kraus et al.,1998), murine epididymal retinoic acid-binding gene (Lareyre et al.,1999), human CD4 (Zhao-Emonet et al., 1998), mouse alpha2 (XI) collagen(Tsumaki, et al., 1998), D1A dopamine receptor gene (Lee, et al., 1997),insulin-like growth factor II (Wu et al., 1997), human plateletendothelial cell adhesion molecule-1 (Almendro et al., 1996).

[0053] B. Termination Signals

[0054] The LEEs and CEEs of the present invention will generallycomprise at least one termination signal. A terminator is comprised ofthe DNA sequences involved in specific termination of an RNA transcriptby an RNA polymerase. Thus, in certain embodiments a termination signalthat ends the production of an RNA transcript is contemplated. However,in eukaryotic systems, the terminator region may also comprise specificDNA sequences that permit site-specific cleavage of the new transcriptso as to expose a polyadenylation site. This signals a specializedendogenous polymerase to add a stretch of about 200 A residues (polyA)to the 3′ end of the transcript. RNA molecules modified with thispolyAtail appear to more stable and are translated more efficiently.Thus, in other embodiments involving eukaryotes, it is preferred thatthat terminator comprises a signal for the cleavage of the RNA, and itis more preferred that the terminator signal promotes polyadenylation ofthe message.

[0055] Terminators contemplated for use in the invention include anyknown terminator of transcription described herein or known to one ofordinary skill in the art, including but not limited to, for example,the termination sequence and polyadenylation site of human growthhormone; termination sequences of house keeping genes including but notlimited to actin or tubulin; or viral termination sequences. Includingbut not limited to eukaryotic viruses such as SV40, MMTV, Polyoma orHIV; or a lack of transcribable or translatable sequence, such as due toa sequence truncation.

[0056] C. Additional Components of LEES and CEES

[0057] The LEEs and CEEs may further comprise at least one additionalregulatory sequence element involved with gene expression, or at leastone additional sequence element to aid LEE or CEE construction oranalysis. These additional elements may enhance or pause the expressionand/or translation of the LEE or CEE sequence(s), enhance theimmunogenicity of the gene-product, direct the gene productintracellularly, or aid in the preparation or analysis of the LEE orCEE. The additional sequence element may include, but is not limited to,at least one initiation signal, at least one internal ribosome bindingsite, at least one multiple cloning site, at least one splicing site, atleast one marker, such as a selectable or screenable marker, or anycombination thereof. The additional elements may be comprised in the 5′sequences and/or 3′ sequences added to the ORF in the construction ofthe LEE or CEE.

[0058] 1. Initiation Signals and Internal Ribosome Binding Sites

[0059] In certain embodiments, the LEE or CEE may comprise at least oneinitiation signal and/or at least one internal ribosome binding site. Inspecific aspects, a specific initiation signal may be required forefficient translation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

[0060] In certain embodiments of the invention, the use of internalribosome entry sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′ methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). IRESelements from two members of the picornavirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message (see U.S. Pat.Nos. 5,925,565 and 5,935,819, herein incorporated by reference).

[0061] 2. Multiple Cloning Sites

[0062] In certain embodiments, the LEE or CEE may comprise at least onemultiple cloning site. A multiple cloning site (MCS) is a nucleic acidregion that contains multiple restriction enzyme sites, any of which canbe used in conjunction with standard recombinant technology to digestthe constuct. (See Carbonelli et al., 1999, Levenson et al., 1998, andCocea, 1997, incorporated herein by reference.) “Restriction enzymedigestion” refers to catalytic cleavage of a nucleic acid molecule withan enzyme that functions only at specific locations in a nucleic acidmolecule. Many of these restriction enzymes are commercially available.Use of such enzymes is widely understood by those of skill in the art.Frequently, a constuct is linearized or fragmented using a restrictionenzyme that cuts within the MCS to enable exogenous sequences to beligated to the construct. “Ligation” refers to the process of formingphosphodiester bonds between two nucleic acid fragments, which may ormay not be contiguous with each other. Techniques involving restrictionenzymes and ligation reactions are well known to those of skill in theart of recombinant technology. In certain aspects, the LEE or CEE may bedigested to release various sequences, such as contained in the ORF, forsize analysis or sequencing.

[0063] 3. Splicing Sites

[0064] Most transcribed eukaryotic RNA molecules will undergo RNAsplicing to remove introns from the primary transcripts. Constructscontaining genomic eukaryotic sequences may require donor and/oracceptor splicing sites to ensure proper processing of the transcriptfor protein expression. (See Chandler et al., 1997, herein incorporatedby reference.)

[0065] 4. Selectable and Screenable Markers

[0066] In certain embodiments of the invention, the cells contain a LEEor CEE construct of the present invention, a cell may be identified invitro or in vivo by including a marker in the LEE or CEE expressionconstruct. Such markers would confer an identifiable change to the cellpermitting easy identification of cells containing the expressionconstruct. Generally, a selectable marker is one that confers a propertythat allows for selection. A positive selectable marker is one in whichthe presence of the marker allows for its selection, while a negativeselectable marker is one in which its presence prevents its selection.An example of a positive selectable marker is a drug resistance marker.

[0067] Usually the inclusion of a drug selection marker aids in thecloning and identification of transformants, for example, genes thatconfer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocinand histidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP or LacZ, whose basis iscalorimetric analysis; GUS whose basis is fluorescence; and LUC whosebasis is lumenescence, are also contemplated. Alternatively, screenableenzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may be utilized. One of skill inthe art would also know how to employ immunologic markers, possibly inconjunction with FACS analysis. The marker used is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product. Further examples ofselectable and screenable markers are well known to one of skill in theart.

[0068] 5. Polyadenylation Signals

[0069] In eukaryotic expression, one may include at least onetranscriptional terminator and at least one polyadenylation signal toeffect termination and proper polyadenylation of the transcript. Thenature of the terminator is not believed to be crucial to the successfulpractice of the invention, and any such sequence may be employed.Preferred embodiments include the SV40 terminator and/or the bovinegrowth hormone terminator, which are convenient and known to functionwell in various target cells. A terminator is comprised of at least onesequence signal of the end of transcription and may include a cleavagesite that enables at least one polyadenylation siquence to be added.Polyadenylation may inclrease the stability of the transcript or mayfacilitate cytoplasmic transport. A terminator may be necessary in vivoto achieve desirable message levels.

[0070] D. LEE and CEE Vectors

[0071] In certain embodiments, the promoter and terminator sequences ofthe LEE or CEE may be regarded as a type of vector. The term “vector” isused to refer to a carrier nucleic acid molecule into which a nucleicacid sequence can be inserted for introduction into a cell where it canbe expressed. A nucleic acid sequence can be “exogenous,” which meansthat it is foreign to the cell into which the vector is being introducedor that the sequence is homologous to a sequence in the cell but in aposition within the host cell nucleic acid in which the sequence isordinarily not found.

[0072] The term “expression vector” refers to a vector containing anucleic acid sequence coding for at least part of a gene product capableof being transcribed. In some cases, RNA molecules are then translatedinto a protein, polypeptide, or peptide. In other cases, these sequencesare not translated, for example, in the production of antisensemolecules or ribozymes. Expression vectors can contain a variety of“control sequences,” which refer to nucleic acid sequences necessary forthe transcription and possibly translation of an operably linked codingsequence in a particular host organism. In the case of LEEs and CEES,minimal control sequences comprising a promoter and/or a terminator maybe added to the sequence that is to be expressed. In addition to apromoter and terminator, other control sequences that governtranscription and translation, vectors and expression vectors maycontain nucleic acid sequences that serve other functions as well andare described infra.

[0073] E. Open Reading Frames (ORF) of Genes

[0074] The LEE or CEE may comprise at least one open reading frame(ORF). An open reading frame comprises a series of tri-nucleotides thatencode one or more amino acids. An ORF is not interrupted by atermination codon (i.e., TAA, TAG, TGA). ORFs isolated from an organismmay encode an endogenous peptide, polypeptide, protein or non-translatedmRNA message. However, many ORFs do not encode for an endogeneouslytranscribed sequence.

[0075] In preferred aspects of the invention, ORFs are isolated from atleast one organism, or a recombinant vector comprising nucleic acidsfrom the organism. One or more organelles (i.e., mitochondria,chloroplasts), cells, tissues or organisms (including viruses) may be asource for the isolated promoters, open reading frames (i.e., genes),termination sequences and other sequences to be used in the constructionof LEEs and CEEs. Methods of nucleic acid isolation are well known toone of ordinary skill in the art (see Sambrook et al. 1989). In otherembodiments, the ORF can be synthetically built by known gene buildingtechniques (Stemmer et al., 1995).

[0076] For screening a nucleic acid library, the screening or isolationprotocol may utilize nucleotide segments or probes that are designed tohybridize to cDNA or genomic sequences of a desired ORF or surroundingsequence(s). Additionally, antibodies designed to bind to the ORF'sexpressed proteins, polypeptides, or peptides may be used as probes toscreen an appropriate DNA expression library. Alternatively, activityassays may be employed. The operation of such screening protocols arewell known to those of skill in the art and are described in detail inthe scientific literature (Sambrook et al. 1989, incorporated herein byreference). Moreover, as the present invention encompasses the isolationand expression of genomic segments as well as cDNA molecules, it iscontemplated that suitable genomic isolation methods, as known to thosein the art, may also be used.

[0077] As used herein “designed to hybridize” means a sequence selectedfor its likely ability to hybridize to an ORF or surrounding sequence(s)due to the expected homology between the ORF or surrounding sequencesand a related nucleic acid sequence probe or primer. Also included aresegments or probes altered to enhance their ability to hybridize to orbind to an ORF or surrounding sequence(s). Additionally, these regionsof homology also include amino acid sequences of 4 or more consecutiveamino acids selected and/or altered to increase conservation of theamino acid sequences in comparison to the same or similar region ofresidues in the same or related genes in one or more species.

[0078] Such sequences may be used as probes for hybridization oroligonucleotide primers for PCR™. Designing such sequences may involveselection of regions of highly conserved nucleotide sequences betweenvarious species for a particular gene or related genes, relative to thegeneral conservation of nucleotides of the gene or related genes in oneor more species. Comparison of the amino acid sequences conservedbetween one or more species for a particular gene may also be used todetermine a group of 4 or more consecutive amino acids that areconserved relative to the protein encoded by the gene or related genes.The nucleotide probe or primers may then be designed from the region ofthe gene that encodes the conserved sequence of amino acids.

[0079] However, random or semi-random probes and primers may be used tohybridize to or amplify ORFs and/or surrounding sequences. Suchuncharacterized ORFs may be sequenced or screened for function using anytechnique described herein or known to one of ordinary skill in the art.

[0080] The nucleotide and protein, polypeptide and peptide sequences forvarious ORFs, promoters, termination sequences and genes have beenpreviously disclosed, and may be found at computerized databases knownto those of ordinary skill in the art. One such database is the NationalCenter for Biotechnology Information's Genbank and GenPept databases(http://www.ncbi.nlm.nih.gov/). The coding regions for these known genesmay be amplified and/or expressed using the techniques disclosed hereinor by any technique that would be know to those of ordinary skill in theart. Such nucleic or amino acid sequences may be used to screen foradditional related ORFs, and may be used to construct LEEs or CEEs.

[0081] The isolated ORFs may then be operatively associated with apromoter, termination signal, and/or other sequence components to form aLEE or CEE. Expressed LEEs or CEEs may be assayed in vivo or in vitrofor any activity or properties of the transcribed or translatedsequences, using any applicable assay described herein or would be knowto one of skill in the art.

[0082] F. Cells

[0083] In particular embodiments, promoters, ORFs, termination sequencesother sequences may be isolated from at least one organelle, cell,tissue or organism. In other embodiments, at least one LEE or CEE may betransfected into at least one organelle, cell, tissue or organism. Inparticular aspects, the LEE or CEE's open reading frame is transcribed,and in more specific aspects, translated into a protein, polypeptide orpeptide in the at least one organelle, cell, tissue or organism.

[0084] As used herein, the terms “cell,” “cell line,” and “cell culture”may be used interchangeably. All of these term also include theirprogeny, which is any and all subsequent generations. It is understoodthat all progeny may not be identical due to deliberate or inadvertentmutations. In the context of expressing a heterologous nucleic acidsequence, “host cell” refers to a prokaryotic or eukaryotic cell, and itincludes any transformable organism that is capable of expressing aheterologous gene encoded by a vector. A host cell can, and has been,used as a recipient for vectors. A host cell may be “transfected” or“transformed,” which refers to a process by which exogenous nucleic acidis transferred or introduced into the host cell. A transformed cellincludes the primary subject cell and its progeny.

[0085] Host cells may be derived from prokaryotes or eukaryotes,depending upon the desired purpose of the expression of the vectorencoded ORF. Numerous cell lines and cultures are available for use as ahost cell, and they can be obtained through the American Type CultureCollection (ATCC), which is an organization that serves as an archivefor living cultures and genetic materials (www.atcc.org). In certainembodiments, a cell may comprise, but is not limited to, at least oneskin, bone, neuron, axon, cartilage, blood vessel, cornea, muscle,facia, brain, prostate, breast, endometrium, lung, pancreas, smallintestine, blood, liver, testes, ovaries, cervix, colon, skin, stomach,esophagus, spleen, lymph node, bone marrow, kidney, peripheral blood,embryonic or ascite cell, and all cancers thereof. An appropriate hostcan be determined by one of skill in the art based on the vectorbackbone and the desired result. Bacterial cells used as host cells forvector expression include DH5α, JM109BL21, and KC8, as well as a numberof commercially available bacterial hosts such as SURE® Competent Cellsand SOLOPACK™ Gold Cells (STRATAGENE®, La Jolla). Alternatively,bacterial cells such as E. coli LE392 could be used as host cells forphage viruses.

[0086] Examples of eukaryotic host cells for replication and/orexpression of a vector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO,Saos, WelH and PC12. Many host cells from various cell types andorganisms are available and would be known to one of skill in the art.

[0087] Some LEE or CEE vectors may employ control sequences that allowit to be expressed in both prokaryotic and eukaryotic cells. One ofskill in the art would further understand the conditions under which toincubate all of the above described host cells to maintain them. Alsounderstood and known are techniques and conditions that would allowlarge-scale production of vectors, as well as production of the nucleicacids encoded by vectors and their cognate polypeptides, proteins, orpeptides.

[0088] 1. Tissues

[0089] The cell or cells to be transformed with a LEE or CEE may becomprised in a tissue. The tissue may be part of or separated from anorganism. In certain embodiments, a tissue may comprise, but is notlimited to, skin, bone, neuron, axon, cartilage, blood vessel, cornea,muscle, facia, brain, prostate, breast, endometrium, lung, pancreas,small intestine, blood, liver, testes, ovaries, cervix, colon, skin,stomach, esophagus, spleen, lymph node, bone marrow, kidney, peripheralblood, embryonic, ascite tissue, meristematic cells, pollen, leaves,anthers, roots, root tips, silk, flowers, kernels, ears, cobs, husks,stalks, and all cancers thereof.

[0090] 2. Organisms

[0091] In certain embodiments, the cell or tissue may be comprised in atleast one organism. In certain embodiments, the organism may be, but isnot limited to, an eubacteria, an archaea, an eukaryote or a virus (seewebpage http://phylogeny.arizona.edu/tree/phylogeny.html).

[0092] a. Eubacteria

[0093] In certain embodiments, the organism is an eubacteria. Inparticular embodiments, the eubacteria may be, but is not limited to, anaquifecales; a thermotogales; a thermodesulfobacterium; a member of thethermus-deinococcus group; a chloroflecales; a cyanobacteria; afirmicutes; a member of the leptospirillum group; a synergistes; amember of the chlorobium-flavobacteria group; a member of thechlamydia-verrucomicrobia group, including but not limited to averrucomicrobia or a chlamydia; a planctomycetales; a flexistipes; amember of the fibrobacter group; a spirochetes; a proteobacteria,including but not limited to an alpha proteobacteria, a betaproteobacteria, a delta & epsilon proteobacteria or a gammaproteobacteria. In certain aspects, an organelle derived from eubacteriaare contemplated, including a mitochondria or a chloroplast.

[0094] b. Archaea

[0095] In certain embodiments, the organism is an archaea (a.k.a.archaebacteria; e.g., a methanogens, a halophiles, a sulfolobus). Inparticular embodiments, the archaea may be, but is not limited to, akorarchaeota; a crenarchaeota, including but not limited to, athermofilum, a pyrobaculum, a thermoproteus, a sulfolobus, ametallosphaera, an acidianus, a thermodiscus, a igneococcus, athermosphaera, a desulfurococcus, a staphylothermus, a pyrolobus, ahyperthermus or a pyrodictium; or an euryarchaeota, including but notlimited to a halobacteriales, methanomicrobiales, a methanobacteriales,a methanococcales, a methanopyrales, an archeoglobales, athermoplasmales or a thermococcales.

[0096] C. Eukaryotes

[0097] In certain embodiments, the organism is an eukaryote (e.g., aprotist, a plant, a fungi, an animal). In particular embodiments, theeukaryote may be, but is not limited to, a microsporidia, a diplomonad,an oxymonad, a retortamonad, a parabasalid, a pelobiont, an entamoebaeor a mitochondrial eukaryote (e.g., an animal, a plant, a fungi, astramenopiles).

[0098] In certain embodiments, the mitochondrial eukaryote may be, butis not limited to, a metazoa (e.g., an animal), a myxozoa, achoanoflagellate, a fungi (e.g., a mushroom, a mold, a yeast, achytrid), a green plant (e.g., a green algae, a land plant), acryptomonad, an ancyromona, plasmodiophorid, a rhodophyta, a centrohelidheliozoa, a cyanophorid, an alveolate (e.g., a dinoflagellate, asporozoan, a ciliate), a stramenopile (e.g., a brown algae, a diatoms,an oomycete, a chrysophyte), an acantharea, a vampyrellid, athaumatomonad, a telonema, a sticholonche, a spongomonad, aramicristate, a pseudospora, a pseudodendromonad, a phalansterium, aphaeodarean radiolaria, a paramyxea, a luffisphaera, a leucodictyon, akathablepharid, a histiona, a haptophyte, an ebriid, a discocelis, adiphylleia, a eesmothoracid, a cryothecomona, a copromyxid, achlorarachnion, a cercomonad, a caecitellus, an apusomonad, anactinophryid or an acanthamoebae.

[0099] In particular aspects, the eukaryote is a metazoa (e.g., ananimal). In certain aspects, the metazoa may be, but is not limited to,a porifera (e.g., a sponge), a cnidaria (e.g., a jellyfish, an anemone,a coral), a ctenophora (e.g., a comb-jelly), an arthropoda (e.g., aninsect, a spider, a crab), an annelida (e.g., a segmented worm), apogonophora, a vestimentifera, an echiura, a mollusca (e.g., a snail, aclam, a squid), a sipuncula, a nemertea (e.g., a ribbon worm), aplatyhelminthes (e.g., a flatworm), a chordata (e.g., a vertebrate), ahemichordata, a lophophorates, a chaetognatha, an echinodermata (e.g., astarfish, a urchin, a sea cucumber), a pseudocoelomates, a placozoa, amonoblastozoa, rhomobozoa, an orthonectida. In particular facets thevertebrate may be a terrestrial vertebrate (e.g., a frog, a salamander,a caecilian, a reptile, a mammal, a bird) or a non-terrestrialvertebrate (e.g., a sharks, a ray, a sawfish, a chimera, a ray-finnedfish, a lobe-finned fish). In additional facets, the mammal may be amonotremata (e.g., a platypus, an echidna), a multituberculata, amarsupialia (e.g., an opossum, a kangaroo), a palaeoryctoids or aneutheria (e.g., a placental mammal).

[0100] In particular facets the eutheria may be, but is not limited to,an edentata (e.g., an anteater, a sloth, an armadillo), a pholidota(e.g., a pangolin), a lagomorpha (e.g., a rabbits), a glires, a rodentia(e.g., a mouse, a rat, a squirrel, a gopher, a porcupine, a beaver), amacroscelidea (e.g., an elephant shrew), a primates (e.g., a monkey, alemur, a gorilla, a chimp, a human), a scandentia (e.g., a tree shrew),a chiroptera (e.g., a bat), a dermoptera (e.g., a colugo, a flyinglemur), an insectivora (e.g., a shrew, a mole, a hedgehog), a creodonta,a carnivora (e.g., a dog, a cat, a bear, a raccon, a weasel, a mongoose,a hyena), a condylarthra, an artiodactyla (e.g., a pig, a deer, acattle, a goat, a sheep, a hippopotamus, a camel), a cetacea (e.g., awhale, a dolphin, a porpoise), a tubulidentata (e.g., an aardvark), aperissodactyla (e.g., a horse, a tapir, a rhinoceros), a hyracoidea(e.g., a hyrax, a dassy), a sirenia (e.g., a manatee, a dugong, a seacow), a desmostylia, an embrythopoda, or a proboscidea (e.g., anelephant).

[0101] In particular embodiments, eukaryote is a fungi. A fungi may be,but is not limited to, a chytridiomycota (e.g., a water mold, anallomyces), a zygomycota (e.g., a bread mold, a rhizopus, a mucor), abasidiomycota (e.g., a mushroom, a rust, a smut) or an ascomycota (e.g.,a sac fungi, a yeast, a penicillium).

[0102] In certain embodiments, the eukaryote is a green plant. A greenplant may be, but is not limited to, a prasinophytes, a chlorophyceae, atrebouxiophyceae, a ulvophyceae, a chlorokybales, a klebsormidiales, azygnematales, a streptophyta, a charales, a coleochaetales or anembryophytes (e.g., a land plant). In particular facets, theembryophytes may be, but is not limited to, a marchantiomorpha (e.g., aliverwort), an Anthoceromorpha (e.g., a hornwort), a bryopsida (e.g., amoss), a lycopsida (e.g., a lycophyte), an equisetopsida (e.g., ahorsetail, a sphenophyte), a filicopsida (e.g., a fern), a spermatopsida(e.g., a seed plant: a flowering plant, a conifer). In particularaspects, the spermatopsida may be, but is not limited to an angiosperm.An angiosperm may include, but is not limited to, a ceratophyllaceae, anymphaeales, a piperales, an aristolochiales, a monocotyledons, aneudicots, a laurales, a chloranthaceae, a winterales or a magnoliales.

[0103] d. Viruses

[0104] In certain embodiments the organism may be a virus. In particularaspects, the virus may be, but is not limited to, a DNA Virus, includingbut not limited to a ssDNA virus or a dsDNA virus; a DNA RNA revtranscribing virus; a RNA virus, including but not limited to a dsRNAvirus, including but not limited to a −ve stranded ssRNA or a +vestranded ssRNA; or an unassigned virus.

[0105] G. Production of LEES and CEES

[0106] The invention further relates to methods of producing a LEE orCEE. Methods for producing a LEE or CEE will generally compriseobtaining a DNA segment comprising an ORF and linking the ORF to apromoter, terminator, or other molecule to create a LEE or CEE.

[0107] The ORF, promoter, terminator or additional nucleic acid(s) maybe obtained by any method described herein or as would be known to oneof ordinary skill in the art, including nucleic acid amplification orchemical synthesis of nucleic acids. Non-limiting examples of syntheticnucleic acid, particularly a synthetic oligonucleotide, include anucleic acid made by in vitro chemically synthesis usingphosphotriester, phosphite or phosphoramidite chemistry and solid phasetechniques such as described in EP 266,032, incorporated herein byreference, or via deoxynucleoside H-phosphonate intermediates asdescribed by Froehler et al., 1986, and U.S. patent Ser. No. 5,705,629,each incorporated herein by reference. A non-limiting example ofenzymatically produced nucleic acid include one produced by enzymes inamplification reactions such as PCR™ (see for example, U.S. Pat. Nos.4,683,202 and 4,682,195, each incorporated herein by reference), or thesynthesis of oligonucleotides described in U.S. Pat. No. 5,645,897,incorporated herein by reference. A non-limiting example of abiologically produced nucleic acid includes recombinant nucleic acidproduction in living cells, such as recombinant DNA vector production inbacteria (see for example, Sambrook et al. 1989, incorporated herein byreference).

[0108] In certain aspect, the present invention concerns at least onepromoter, terminator, ORF and/or other nucleic acid that is an isolatednucleic acid. As used herein, the term “isolated nucleic acid” refers toat least one nucleic acid molecule that has been isolated free of, or isotherwise free of, the bulk of the total genomic and transcribed nucleicacids of one or more organelles, cells, tissues or organisms. In certainembodiments, “isolated nucleic acid” refers to a nucleic acid that hasbeen isolated free of, or is otherwise free of, bulk of cellularcomponents and macromolecules such as lipids, proteins, small biologicalmolecules, and the like. As different species may have a RNA or a DNAcontaining genome, the term “isolated nucleic acid” encompasses both theterms “isolated DNA” and “isolated RNA”. Thus, the isolated nucleic acidmay comprise a RNA or DNA molecule isolated from, or otherwise free of,the bulk of total RNA, DNA or other nucleic acids of a particularspecies. As used herein, an isolated nucleic acid isolated from aparticular species is referred to as a “species specific nucleic acid.”When designating a nucleic acid isolated from a particular species, suchas human, such a type of nucleic acid may be identified by the name ofthe species. In a non-limiting example, a nucleic acid isolated from oneor more humans would be an “isolated human nucleic acid.”

[0109] The linking of an ORF to a promoter, terminator and/or anadditional molecule (i.e., another nucleic acid), may be a covalent ornon-covalent attachment or association. In a non-limiting example, anon-covalent association contemplated may be simply admixing a promoter,terminator and/or additional nucleic acid with the ORF nucleic acid. Ina more preferred embodiment, one or more of ends of the promoter,terminator, additional molecule and/or ORF comprise complementarynucleic acids that promote annealing of one or more ends of thepromoter, terminator, additional molecule and/or ORF to themselves or toeach other. If one or more of the nucleic acids are double stranded, theend that is complementary may be an overhanging single strand from thedouble stranded molecule. Single-stranded regions of sufficient lengthand/or complementary anneal, and the attached components may be directlydelivered into a cell, tissue or organism.

[0110] In one method that may be employed, a dUMP-containing tail issynthesized at the 5′ end of PCR® primers. Following amplification,uracil DNA glycosylase (UDG) is used to cleave the glycosylic bondbetween the sugar and base. This creates an abasic site whichdestabilizes base-pairing in the double-stranded DNA and creates a 5′single-stranded overhang available for linking.

[0111] LEEs and CEEs may be made by any number of methods other than thedU method described above. For example, there are multiple examples inthe literature of methods of generating overhangs for plasmid cloning.The inventors have used the below-described alternative methods forgenerating the overhangs typically employed in producing LEEs.

[0112] For example, abasic phosphoramidates may be used. PCR® primersare synthesized so as to contain a “base-less” phosphoramidite at aposition where one desires the single-stranded/double-stranded junctionto be on the final PCR® product. This “nucleotide” has no sugar residue,therefore during amplification Taq polymerase stops when it reaches thisposition, leaving a protruding 5′ end. In one drawback of this method,the modified phosphoramidite is unstable and as a result the wholeprimer that is made for this purpose is unstable. A related methodinvolves the use of dSpacer phosphoramidates. These are used in the sameway as abasics: PCR® primers are built that contain a modifiedphosphoramidite at some position where one desires thesingle-stranded/double-stranded junction to be on the final PCR®product. In this case, the stability of the abasic site was improved byinserting a “base-like” structure in position of the base. The syntheticabasic and dSpacer methods tend to be inefficient, in that they requirethe synthetic oligo regions of the PCR® product (the 5′ overhangs) toanneal.

[0113] Methods involving T4 DNA polymerase may be used to createoverhangs. The exonuclease activity of this enzyme is used to digest the3′ ends of PCR® products, resulting in a 5′ single-stranded overhang.The extent of the digestion (and therefore the length of the overhang)is controlled by designing a primer with a specific sequence that lacksone of the four nucleotide bases from the 5′ end until the desireddouble/single-stranded junction point. Only the nucleotide to pair withthis position is provided during digestion.

[0114] One can use rU/RNase A to create LEEs. This method isconceptually similar to the dU/UDG method. However, instead of a DNA(dU) version of uracil, one can use the RNA molecule (rU) to incorporateinto PCR® primers. These primers were used for standard PCR®amplification. To generate the overhangs one simply exposes the PCR®product to RNaseA, which is far cheaper than UDG. This method works wellbut has a drawback in the instability of RNA-containing primers relativeto completely DNA primers.

[0115] Methods involving long/short PCR® priming are useful to prepareLEEs. One can design two primers for one side of a PCR® product, withone sitting down on the template 12-15 nucleotides upstream of theother. Amplification with both primers in addition to the usual one atthe other end generated three types of annealed products: 2 longs (25%),2 shorts (25%), and 1 long/1 short (50% of the population). Thelong/short products are the useful products with single-strandedoverhangs.

[0116] With appropriate modifications, each of these technologies can beemployed to make CEEs as well as LEEs.

[0117] To enable annealing of single stranded ends, the ends arepreferably complementary or semi-complementary. Designing complementaryor semi-complementary sequences of nucleic acids are well know to one ofordinary skill in the art. Nucleic acid(s) that are “complementary” or“complement(s)” are those that are capable of base-pairing according tothe standard Watson-Crick, Hoogsteen or reverse Hoogsteen bindingcomplementarity rules. As used herein, the term “complementary” or“complement(s)” also refers to nucleic acid(s) that are substantiallycomplementary, as may be assessed by the same nucleotide comparison setforth above. The term “substantially complementary” refers to a nucleicacid comprising at least one sequence of consecutive nucleobases, orsemiconsecutive nucleobases if one or more nucleobase moieties are notpresent in the molecule, that are capable of hybridizing or annealing toat least one nucleic acid strand or duplex even if less than allnucleobases do not base pair with a counterpart nucleobase. In certainembodiments, a “substantially complementary” nucleic acid contains atleast one sequence in which about 70%, about 71%, about 72%, about 73%,about 74%, about 75%, about 76%, about 77%, about 77%, about 78%, about79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%,about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,about 99%, to about 100%, and any range therein, of the nucleobasesequence is capable of base-pairing with at least one single or doublestranded nucleic acid molecule during hybridization or annealing. Incertain embodiments, the term “substantially complementary” refers to atleast one nucleic acid that may hybridize to at least one nucleic acidstrand or duplex in stringent conditions. In certain embodiments, a“partly complementary” nucleic acid comprises at least one sequence thatmay hybridize in low stringency conditions to at least one single ordouble stranded nucleic acid, or contains at least one sequence in whichless than about 70% of the nucleobase sequence is capable ofbase-pairing with at least one single or double stranded nucleic acidmolecule during hybridization or annealing.

[0118] In certain embodiments, the complementary or semi-complementaryends of the ORF, promoter, terminator and/or additional molecule promotenon-covalent association of their ends, in any combination desired ordesigned by a skilled practitioner. Thus, linear or circularconformations of the ORF, promoter, terminator and/or additionalmolecule are contemplated through non-covalent associations. In otheraspects, the semi-complementary or complementary ends may promoteenzymatic ligation. Of course, one or more ends of an ORF, promoter,terminator and/or additional molecule may be designed to promoteannealing to form linear or circular elements. Alternatively, thepromoter, terminator, ORF and/or additional molecule may besynthetically or biologically produced as one nucleic acid.

[0119] In a preferred embodiment, one or more ORFs would be amplified,and then be linked (i.e., associated, annealed, and/or ligated) to astandard promoter and/or terminator. For example, most gene screeningassays will require fusing a common promoter and/or terminator to avariety of ORFs, or a reporter ORF to a variety of promoters and/orterminators. In a non-limiting example, screening genes in mammaliancells would require fusing the ORFs to a eukaryotic promoter andterminator. Therefore, an ideal LEE system will typically involveamplification of only an ORF which would then be linked to a set ofstandard promoter and/or terminator sequences. In certain embodiments,the promoter, terminator and/or ORF are produced via amplification asone unit.

[0120] In additional embodiments, it is not required for allapplications that a terminator be provided. For instance, in methodsinvolving cell-free expression, no terminator may be required.

[0121] Though not required, a nucleic acid, such as a promoter, ORF,terminator, LEE or CEE may be purified on polyacrylamide gels, cesiumchloride centrifugation gradients, filter gradient or by any other meansknown to one of ordinary skill in the art (see for example, Sambrook etal. 1989, incorporated herein by reference).

[0122] H. Methods of LEE and CEE Delivery

[0123] Suitable methods for LEE or CEE delivery for transformation of aorganelle, cell, tissue or organim for use with the current inventionare believed to include virtually any method by which DNA can beintroduced into a cell, such as by direct delivery of DNA such as byPEG-mediated transformation of protoplasts (Omirulleh et al., 1993), bydesiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985), byelectroporation (U.S. Pat. No. 5,384,253, specifically incorporatedherein by reference in its entirety), by agitation with silicon carbidefibers (Kaeppler et al., 1990; U.S. Pat. No. 5,302,523, specificallyincorporated herein by reference in its entirety; and U.S. Pat. No.5,464,765, specifically incorporated herein by reference in itsentirety), by Agrobacterium-mediated transformation (U.S. Pat. Nos.5,591,616 and 5,563,055; both specifically incorporated herein byreference) and by acceleration of DNA coated particles (U.S. Pat. Nos.5,550,318; 5,538,877; and 5,538,880; each specifically incorporatedherein by reference in its entirety), etc. Through the application oftechniques such as these, organelle(s), cell(s), tissue(s) ororganism(s) may be stably or transciently transformed. In certainembodiments, acceleration methods are preferred and include, forexample, microprojectile bombardment and the like.

[0124] 1. Injection

[0125] In certain embodiments, the LEE or CEE may be delivered via oneor more injections, for example, either subcutaneously, intradermally orintramuscularly.

[0126] The data in FIG. 7A and FIG. 7B show that PCR®-generated LEEs canbe used with other transfection protocols such as an injection (i.e., aneedle injection). When the supercoiled, replicative control plasmid andLEE vectors were introduced intramuscularly (i.m.) by needle injectionin a saline solution, the LUC activity encoded by the noncovalentlylinked LEE was 15% of that produced by the LUC plasmid. Addition ofligase to the LEE prior to injection raised LUC activity to 41% of theplasmid standard (FIG. 7B). By contrast, addition of ligase did notfurther improve expression from LEEs introduced with the gene gun, whichwas similar to the plasmid standard (FIG. 7A). These results areconsistent with the observation that the gene-gun delivers samplesdirectly inside skin cells (Williams et al., 1991) while a needleintroduces DNA into the extracellular space of muscle tissue (Wolff etal., 1990), where exonucleases are prevalent.

[0127] 2. Electroporation

[0128] In certain embodiments of the present invention, the LEE or CEEis introduced into the cell via electroporation. Electroporationinvolves the exposure of a suspension of cells and DNA to a high-voltageelectric discharge. Where one wishes to introduce DNA by means ofelectroporation, it is contemplated that the method of Krzyzek et al.(U.S. Pat. No. 5,384,253, incorporated herein by reference in itsentirety) will be particularly advantageous. In this method, certaincell wall-degrading enzymes, such as pectin-degrading enzymes, areemployed to render the target recipient cells more susceptible totransformation by electroporation than untreated cells. Alternatively,recipient cells are made more susceptible to transformation bymechanical wounding.

[0129] Transfection of eukaryotic cells using electroporation has beenquite successful. Mouse pre-B lymphocytes have been transfected withhuman kappa-immunoglobulin genes (Potter et al., 1984), and rathepatocytes have been transfected with the chloramphenicolacetyltransferase gene (Tur-Kaspa et al., 1986) in this manner.

[0130] To effect transformation by electroporation in cells such as, butnot limited to, plant cells, one may employ either friable tissues, suchas a suspension culture of cells or embryogenic callus or alternativelyone may transform immature embryos or other organized tissue directly.In this technique, one would partially degrade the cell walls of thechosen cells by exposing them to pectin-degrading enzymes (pectolyases)or mechanically wounding in a controlled manner. Examples of somespecies which have been transformed by electroporation of intact cellsinclude maize (U.S. Pat. No. 5,384,253; Rhodes et al., 1995; D'Halluinet al., 1992), wheat (Zhou et al., 1993), tomato (Hou and Lin, 1996),soybean (Christou et al., 1987) and tobacco (Lee et al., 1989).

[0131] One also may employ protoplasts for electroporationtransformation of plants (Bates, 1994; Lazzeri, 1995). For example, thegeneration of transgenic soybean plants by electroporation ofcotyledon-derived protoplasts is described by Dhir and Widholm in Intl.Patent Appl. Publ. No. WO 9217598 (specifically incorporated herein byreference). Other examples of species for which protoplasttransformation has been described include barley (Lazerri, 1995),sorghum (Battraw et al., 1991), maize (Bhattachaijee et al., 1997),wheat (He et al., 1994) and tomato (Tsukada, 1989).

[0132] 3. Microprojectile Bombardment

[0133] Microprojectile bombardment techniques are a preferred method ofintroducing CEEs and LEEs into at least one, organelle, cell, tissue ororganism (U.S. Pat. Nos. 5,550,318; 5,538,880; 5,610,042; and PCTApplication WO 94/09699; each of which is specifically incorporatedherein by reference in its entirety). There are a wide variety ofmicroprojectile bombardment techniques known in the art, many of whichare applicable to the invention. However, the inventors have used thegeneral protocol described in the specific examples. In some of thestudies described herein the specific examples, genes were introducedinto skin cells with a gene gun (Examples 2, 3, 4, 9, 10, 11 and 16).

[0134] In this method, particles may be coated with nucleic acids anddelivered into cells by a propelling force. Exemplary particles includethose comprised of tungsten, platinum, and preferably, gold. It iscontemplated that in some instances DNA precipitation onto metalparticles would not be necessary for DNA delivery to a recipient cellusing microprojectile bombardment. However, it is contemplated thatparticles may contain DNA rather than be coated with DNA. Hence, it isproposed that DNA-coated particles may increase the level of DNAdelivery via particle bombardment but are not, in and of themselves,necessary.

[0135] For the bombardment, cells in suspension are concentrated onfilters or solid culture medium. Alternatively, immature embryos orother target cells may be arranged on solid culture medium. The cells tobe bombarded are positioned at an appropriate distance below themacroprojectile stopping plate.

[0136] An illustrative embodiment of a method for delivering DNA intocells, including but not limited to plant cells, by acceleration is theBiolistics Particle Delivery System, which can be used to propelparticles coated with DNA or cells through a screen, such as a stainlesssteel or Nytex screen, onto a filter surface covered with cells, such asfor example, a monocot plant cells cultured in suspension. The screendisperses the particles so that they are not delivered to the recipientcells in large aggregates. It is believed that a screen interveningbetween the projectile apparatus and the cells to be bombarded reducesthe size of projectiles aggregate and may contribute to a higherfrequency of transformation by reducing the damage inflicted on therecipient cells by projectiles that are too large.

[0137] Microprojectile bombardment techniques are widely applicable, andmay be used to transform virtually various cells, tissues or organism,such as for example any plant species. Examples of species for whichhave been transformed by microprojectile bombardment include monocotspecies such as maize (PCT Application WO 95/06128), barley (Ritala etal., 1994; Hensgens et al., 1993), wheat (U.S. Pat. No. 5,563,055,specifically incorporated herein by reference in its entirety), rice(Hensgens et al., 1993), oat (Torbet et al., 1995; Torbet et al., 1998),rye (Hensgens et al., 1993), sugarcane (Bower et al., 1992), and sorghum(Casas et al., 1993; Hagio et al., 1991); as well as a number of dicotsincluding tobacco (Tomes et al., 1990; Buising and Benbow, 1994),soybean (U.S. Pat. No. 5,322,783, specifically incorporated herein byreference in its entirety), sunflower (Knittel et al. 1994), peanut(Singsit et al., 1997), cotton (McCabe and Martinell, 1993), tomato(VanEck et al. 1995), and legumes in general (U.S. Pat. No. 5,563,055,specifically incorporated herein by reference in its entirety).

[0138] 4. Liposome-Mediated Transfection

[0139] In a further embodiment of the invention, a LEE or CEE may beentrapped in a liposome. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). Also contemplated is an expression construct complexedwith Lipofectamine (Gibco BRL) or Superfect (Qiagen).

[0140] Liposome-mediated nucleic acid delivery and expression of foreignDNA in vitro has been very successful (Nicolau and Sene, 1982; Fraley etal., 1979; Nicolau et al., 1987). Wong et al. (1980) demonstrated thefeasibility of liposome-mediated delivery and expression of foreign DNAin cultured chick embryo, HeLa and hepatoma cells.

[0141] In certain embodiments of the invention, the liposome may becomplexed with a hemagglutinating virus (HVJ). This has been shown tofacilitate fusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments,the liposome may be complexed or employed in conjunction with nuclearnon-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, the liposome may be complexed or employed inconjunction with both HVJ and HMG-1. In other embodiments, the deliveryvehicle may comprise a ligand and a liposome.

[0142] 5. Calcium Phosphate or DEAE-Dextran

[0143] In other embodiments of the present invention, the LEE or CEE isintroduced to the cells using calcium phosphate precipitation. Human KBcells have been transfected with adenovirus 5 DNA (Graham and Van DerEb, 1973) using this technique. Also in this manner, mouse L(A9), mouseC127, CHO, CV-1, BHK, NIH3T3 and HeLa cells were transfected with aneomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes weretransfected with a variety of marker genes (Rippe et al., 1990).

[0144] In another embodiment, the LEE or CEE is delivered into the cellusing DEAE-dextran followed by polyethylene glycol. In this manner,reporter plasmids were introduced into mouse myeloma and erythroleukemiacells (Gopal, 1985).

[0145] 6. Direct Microinjection or Sonication Loading

[0146] Further embodiments of the present invention include theintroduction of the LEE or CEE by direct microinjection or sonicationloading. Direct microinjection has been used to introduce nucleic acidconstructs into Xenopus oocytes (Harland and Weintraub, 1985), and LTK⁻fibroblasts have been transfected with the thymidine kinase gene bysonication loading (Fechheimer et al., 1987).

[0147] 7. Receptor Mediated Transfection

[0148] Still further, LEEs or CEEs that may be employed to delivernucleic acid constructs to target cells are receptor-mediated deliveryvehicles. These take advantage of the selective uptake of macromoleculesby receptor-mediated endocytosis that will be occurring in the targetcells. In view of the cell type-specific distribution of variousreceptors, this delivery method adds another degree of specificity tothe present invention. Specific delivery in the context of anothermammalian cell type is described by Wu and Wu (1993; incorporated hereinby reference).

[0149] Certain receptor-mediated gene targeting vehicles comprise a cellreceptor-specific ligand and a DNA-binding agent. Others comprise a cellreceptor-specific ligand to which the DNA construct to be delivered hasbeen operatively attached. Several ligands have been used forreceptor-mediated gene transfer (Wu and Wu, 1987; Wagner et al., 1990;Perales et al., 1994; Myers, EPO 0273085), which establishes theoperability of the technique. In certain aspects of the presentinvention, the ligand will be chosen to correspond to a receptorspecifically expressed on the EOE target cell population.

[0150] In other embodiments, the DNA delivery vehicle component of acell-specific gene targeting vehicle may comprise a specific bindingligand in combination with a liposome. The nucleic acids to be deliveredare housed within the liposome and the specific binding ligand isfunctionally incorporated into the liposome membrane. The liposome willthus specifically bind to the receptors of the target cell and deliverthe contents to the cell. Such systems have been shown to be functionalusing systems in which, for example, epidermal growth factor (EGF) isused in the receptor-mediated delivery of a nucleic acid to cells thatexhibit upregulation of the EGF receptor.

[0151] In still further embodiments, the DNA delivery vehicle componentof the targeted delivery vehicles may be a liposome itself, which willpreferably comprise one or more lipids or glycoproteins that directcell-specific binding. For example, Nicolau et al. (1987) employedlactosyl-ceramide, a galactose-terminal asialganglioside, incorporatedinto liposomes and observed an increase in the uptake of the insulingene by hepatocytes. It is contemplated that the tissue-specifictransforming constructs of the present invention can be specificallydelivered into the target cells in a similar manner.

[0152] 8. Plant Directed Transformation Techniques

[0153] a. Agrobacterium-Mediated Transformation

[0154] Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art. See, for example, the methods described by Fraley etal., (1985), Rogers et al., (1987) and U.S. Pat. No. 5,563,055,specifically incorporated herein by reference in its entirety.

[0155] Agrobacterium-mediated transformation is most efficient indicotyledonous plants and is the preferable method for transformation ofdicots, including Arabidopsis, tobacco, tomato, and potato. Indeed,while Agrobacterium-mediated transformation has been routinely used withdicotyledonous plants for a number of years, it has only recently becomeapplicable to monocotyledonous plants. Advances inAgrobacterium-mediated transformation techniques have now made thetechnique applicable to nearly all monocotyledonous plants. For example,Agrobacterium-mediated transformation techniques have now been appliedto rice (Hieietal., 1997; Zhang et al., 1997; U.S. Pat. No. 5,591,616,specifically incorporated herein by reference in its entirety), wheat(McCormac et al., 1998), barley (Tingay et al., 1997; McCormac et al.,1998), and maize (Ishidia et al., 1996).

[0156] Modem Agrobacterium transformation vectors are capable ofreplication in E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate the construction of vectors capable ofexpressing various polypeptide coding genes. The vectors described(Rogers et al., 1987) have convenient multi-linker regions flanked by apromoter and a polyadenylation site for direct expression of insertedpolypeptide coding genes and are suitable for present purposes. Inaddition, Agrobacterium containing both armed and disarmed Ti genes canbe used for the transformations. In those plant strains whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene transfer.

[0157] b. Other Transformation Methods

[0158] Transformation of plant protoplasts can be achieved using methodsbased on calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, e.g.,Potrykus et al., 1985; Lorz et al., 1985; Omirulleh et al., 1993; Frommet al., 1986; Uchimiya et al., 1986; Callis et al., 1987; Marcotte etal., 1988).

[0159] Application of these systems to different plant strains dependsupon the ability to regenerate that particular plant strain fromprotoplasts. Illustrative methods for the regeneration of cereals fromprotoplasts have been described (Fujimara et al., 1985; Toriyama et al.,1986; Yamada et al., 1986; Abdullah et al., 1986; Omirulleh et al., 1993and U.S. Pat. No. 5,508,184; each specifically incorporated herein byreference in its entirety). Examples of the use of direct uptaketransformation of cereal protoplasts include transformation of rice(Ghosh-Biswas et al., 1994), sorghum (Battraw and Hall, 1991), barley(Lazerri, 1995), oat (Zheng and Edwards, 1990) and maize (Omirulleh etal., 1993).

[0160] To transform plant strains that cannot be successfullyregenerated from protoplasts, other ways to introduce DNA into intactcells or tissues can be utilized. For example, regeneration of cerealsfrom immature embryos or explants can be effected as described (Vasil,1989). Also, silicon carbide fiber-mediated transformation may be usedwith or without protoplasting (Kaeppler, 1990; Kaeppler et al., 1992;U.S. Pat. No. 5,563,055, specifically incorporated herein by referencein its entirety). Transformation with this technique is accomplished byagitating silicon carbide fibers together with cells in a DNA solution.DNA passively enters as the cell are punctured. This technique has beenused successfully with, for example, the monocot cereals maize (PCTApplication WO 95/06128, specifically incorporated herein by referencein its entirety; Thompson, 1995) and rice (Nagatani, 1997).

[0161] I. Non-Protein-Expressing Sequences

[0162] In certain embodiments, the LEE or CEE may express messages thatare not translated. DNA may be introduced into organisms for the purposeof expressing RNA transcripts that function to affect phenotype yet arenot translated into protein. Two examples are antisense RNA and RNA withribozyme activity. Both may serve possible functions in reducing oreliminating expression of native or introduced genes. However, asdetailed below, DNA need not be expressed to effect the phenotype of anorganism.

[0163] 1. Antisense RNA

[0164] In certain aspects, a LEE or CEE may express an antisensemessage. ORFs, particularly those from genes may be constructed orisolated, which when transcribed, produce antisense RNA that iscomplementary to all or part(s) of a targeted messenger RNA(s). Theantisense RNA reduces production of the polypeptide product of themessenger RNA. The polypeptide product may be any protein encoded by thecell's genome. The aforementioned genes will be referred to as antisensegenes. An antisense gene may thus be introduced into a cell bytransformation methods to produce a novel transgenic cell or organismwith reduced expression of a selected protein of interest. For example,the protein may be an enzyme that catalyzes a reaction in the cell ororganism. Reduction of the enzyme activity may reduce or eliminateproducts of the reaction which include any enzymatically synthesizedcompound in the cell or organism such as fatty acids, amino acids,carbohydrates, nucleic acids and the like.

[0165] Alternatively, in a non-limiting example such as thetransformation of a plant cell, the protein may be a storage protein,such as a zein, or a structural protein, the decreased expression ofwhich may lead to changes in seed amino acid composition or plantmorphological changes respectively. The possibilities cited above areprovided only by way of example and do not represent the full range ofapplications.

[0166] 2. Ribozymes

[0167] In other aspects, the LEE or CEE may produce a ribozyme. ORFs maybe constructed or isolated which, when transcribed, produce RNA enzymes(ribozymes) that can act as endoribonucleases and catalyze the cleavageof RNA molecules with selected sequences. The cleavage of selectedmessenger RNAs can result in the reduced production of their encodedpolypeptide products. These genes may be used to prepare novel one ormore cells, tissues and organisms which possess them. The transgeniccells, tissues or organisms may possess reduced levels of polypeptidesincluding, but not limited to, the polypeptides cited above.

[0168] Ribozymes are RNA-protein complexes that cleave nucleic acids ina site-specific fashion. Ribozymes have specific catalytic domains thatpossess endonuclease activity (Kim and Cech, 1987; Gerlach et al., 1987;Forster and Symons, 1987). For example, a large number of ribozymesaccelerate phosphoester transfer reactions with a high degree ofspecificity, often cleaving only one of several phosphoesters in anoligonucleotide substrate (Cech et al., 1981; Michel and Westhof, 1990;Reinhold-Hurek and Shub, 1992). This specificity has been attributed tothe requirement that the substrate bind via specific base-pairinginteractions to the internal guide sequence (“IGS”) of the ribozymeprior to chemical reaction.

[0169] Ribozyme catalysis has primarily been observed as part ofsequence-specific cleavage/ligation reactions involving nucleic acids(Joyce, 1989; Cech et al., 1981). For example, U.S. Pat. No. 5,354,855reports that certain ribozymes can act as endonucleases with a sequencespecificity greater than that of known ribonucleases and approachingthat of the DNA restriction enzymes.

[0170] Several different ribozyme motifs have been described with RNAcleavage activity (Symons, 1992). Examples include sequences from theGroup I self splicing introns including Tobacco Ringspot Virus (Prody etal., 1986), Avocado Sunblotch Viroid (Palukaitis et al., 1979), andLucerne Transient Streak Virus (Forster and Symons, 1987). Sequencesfrom these and related viruses are referred to as hammerhead ribozymebased on a predicted folded secondary structure.

[0171] Other suitable ribozymes include sequences from RNase P with RNAcleavage activity (Yuan et al., 1992, Yuan and Altman, 1994, U.S. Pat.Nos. 5,168,053 and 5,624,824), hairpin ribozyme structures(Berzal-Herranz et al., 1992; Chowrira et al., 1993) and Hepatitis Deltavirus based ribozymes (U.S. Pat. No. 5,625,047). The general design andoptimization of ribozyme directed RNA cleavage activity has beendiscussed in detail (Haseloff and Gerlach, 1988, Symons, 1992, Chowriraet al., 1994; Thompson et al., 1995).

[0172] The other variable on ribozyme design is the selection of acleavage site on a given target RNA. Ribozymes are targeted to a givensequence by virtue of annealing to a site by complimentary base pairinteractions. Two stretches of homology are required for this targeting.These stretches of homologous sequences flank the catalytic ribozymestructure defined above. Each stretch of homologous sequence can vary inlength from 7 to 15 nucleotides. The only requirement for defining thehomologous sequences is that, on the target RNA, they are separated by aspecific sequence which is the cleavage site. For hammerhead ribozyme,the cleavage site is a dinucleotide sequence on the target RNA is auracil (U) followed by either an adenine, cytosine or uracil (A,C or U)(Perriman et al., 1992; Thompson et al., 1995). The frequency of thisdinucleotide occurring in any given RNA is statistically 3 out of 16.Therefore, for a given target messenger RNA of 1000 bases, 187dinucleotide cleavage sites are statistically possible.

[0173] Designing and testing ribozymes for efficient cleavage of atarget RNA is a process well known to those skilled in the art. Examplesof scientific methods for designing and testing ribozymes are describedby Chowrira et al., (1994) and Lieber and Strauss (1995), eachincorporated by reference. The identification of operative and preferredsequences for use in down regulating a given gene is simply a matter ofpreparing and testing a given sequence, and is a routinely practiced“screening” method known to those of skill in the art.

[0174] 3. Induction of Gene Silencing

[0175] In additional aspects, the LEE or CEE may be transcribed topromote gene silencing. It also is possible that ORFs derived from genesmay be introduced to produce novel cells, tissues and organisms whichhave reduced expression of a native gene product by the mechanism ofco-suppression. It has been demonstrated in tobacco, tomato, and petunia(Goring et al., 1991; Smith et al., 1990; Napoli et al., 1990; van derKrol et al., 1990) that expression of the sense transcript of a nativegene will reduce or eliminate expression of the native gene in a mannersimilar to that observed for antisense genes. The introduced gene mayencode all or part of the targeted native protein but its translationmay not be required for reduction of levels of that native protein.

[0176] 4. Non-RNA-Expressing Sequences

[0177] In further embodiments, LEE or CEEs may be used to tag a cell,tissue or organism, or mutate a gene. DNA elements including those oftransposable elements such as Ds, Ac, or Mu, may be inserted into a geneto cause mutations. These DNA elements may be inserted in order toinactivate (or activate) a gene and thereby “tag” a particular trait. Inthis instance the transposable element does not cause instability of thetagged mutation, because the utility of the element does not depend onits ability to move in the genome. Once a desired trait is tagged, theintroduced DNA sequence may be used to clone the corresponding gene,e.g., using the introduced DNA sequence as a PCR primer together withPCR gene cloning techniques (Shapiro, 1983; Dellaporta et al., 1988).Once identified, the entire gene(s) for the particular trait, includingcontrol or regulatory regions where desired, may be isolated, cloned andmanipulated as desired. The utility of DNA elements introduced into anorganism for purposes of gene tagging is independent of the DNA sequenceand does not depend on any biological activity of the DNA sequence,i.e., transcription into RNA or translation into protein. The solefunction of the DNA element is to disrupt the DNA sequence of a gene.

[0178] It is contemplated that unexpressed DNA sequences, includingnovel synthetic sequences, could be introduced into cells, tissues andorganims as proprietary “labels” of those cells, tissues and organisms,particularly plants and seeds thereof. It would not be necessary for alabel DNA element to disrupt the function of a gene endogenous to thehost organism, as the sole function of this DNA would be to identify theorigin of the cell, tissue or organism. For example, one could introducea unique DNA sequence into a plant and this DNA element would identifyall cells, plants, and progeny of these cells as having arisen from thatlabeled source. It is proposed that inclusion of label DNAs would enableone to distinguish proprietary germplasm or germplasm derived from such,from unlabelled germplasm.

[0179] Another possible element which may be introduced is a matrixattachment region element (MAR), such as the chicken lysozyme A element(Stief, 1989), which can be positioned around an expressible gene ofinterest to effect an increase in overall expression of the gene anddiminish position dependent effects upon incorporation into the genome,particularly a plant genome (Stief et al., 1989; Phi-Van et al., 1990).

[0180] J. Exogenous Genes for Modification of Plant Phenotypes

[0181] A particularly important advance of the present invention is thatit provides methods and compositions for the efficient expression ofselected proteins in plant cells. LEE and CEE constructs may be madewith various plant specific promoters. Promoters for plant specificexpression are known to those of skill in the art, and include but arenot limited to, any constitutive, inducible, tissue or organ specific,or developmental stage specific promoter which can be expressed in theparticular plant cell. Suitable such promoters are disclosed in Weisinget al, supra.

[0182] Promoters suitable for use herein include, but are not limitedto, at least one regulatory sequence from the T-DNA of A. tumefaciens,including mannopine synthase, nopaline synthase, and octopine synthase;alcohol dehydrogenase promoter from corn; light inducible promoters suchas ribulose-biphosphate-carboxylase small subunit gene from a variety ofspecies and the major chlorophyll a/b binding protein gene promoter;histone promoters (EP 507 698), actin promoters; maize ubiquitin 1promoter (Christensen et al. (1996) Transgenic Res. 5:213); 35S and 19Spromoters of cauliflower mosaic virus; developmentally regulatedpromoters such as the waxy, zein, or bronze promoters from maize; aswell as synthetic or other natural promoters which are either inducibleor constitutive, including those promoters exhibiting organ specificexpression or expression at specific development stage(s) of the plant,like the alpha-tubulin promoter disclosed in U.S. Pat. No. 5,635,618.

[0183] Other elements such as introns, enhancers, termination sequencesand the like, may also be present in the LEE or CEE. These elements mustbe compatible with the remainder of the LEE or CEE gene constructions.Such elements may or may not be necessary for the function of the gene,although they may provide a better expression or functioning of the geneby effecting transcription, stability of the mRNA, or the like. Suchelements may be included in the nucleic acid as desired to obtain theoptimal performance of the transforming gene in the organism, such asbut not limited to a plant. In a non-limiting example, the maize Adh1Sfirst intron maybe placed between the promoter and the coding sequenceof a particular heterologous nucleic acid. This intron, when included ina gene construction, is known to generally increase expression in maizecells of a protein. (Callis et al. (1987) Genes Dev. 1:1183). Othersuitable introns include, but are not limited to, at least one firstintron of the shrunken-i gene of maize (Maas et al. (1991) Plant Mol.Biol. 16:199); the first intron of the castor bean catalase (cat-1) gene(Ohta et al. (1990) Plant Cell Physiol. 31:805); potato catalase secondintron of the ST-LSI gene (Vancanneyt et al. (1990) Mol. Gen. Genet.220:245); tobacco yellow dwarf virus DSV intron (Morris et al. (1992)Virology 187:633; actin-1 (act-1) intron from rice (McElroy et al.(1990) Plant Cell 2:163); and triose phosphate isomerase (TPI) intron 1(Snowden et al. (1996) Plant Mol. Biol. 31:689). However, sufficientexpression for a gene to perform satisfactorily can often by obtainedwithout an intron. (Battraw et al. (1990) Plant Mol. Biol. 15:527).

[0184] The LEE or CEE construct comprising the heterologous nucleic acidmay also comprise sequences coding for a transit peptide, to drive theprotein coded by the heterologous gene into an organelle (e.g., achloroplast) of the plant cells. Such transit peptides are well known tothose of ordinary skill in the art, and may include single transitpeptides, as well as multiple transit peptides obtained by thecombination of sequences coding for at least two transit peptides. Onetransit peptide is the Optimized Transit Peptide disclosed in U.S. Pat.No. 5,635,618, comprising in the direction of transcription a first DNAsequence encoding a first chloroplast transit peptide, a second DNAsequence encoding an N-terminal domain of a mature protein naturallydriven into the chloroplasts, and a third DNA sequence encoding a secondchloroplast transit peptide.

[0185] The choice of a selected protein for expression in a plant hostcell in accordance with the invention will depend on the purpose of thetransformation. One of the major purposes of transformation of cropplants is to add commercially desirable, agronomically important traitsto the plant. Such traits include, but are not limited to, herbicideresistance or tolerance; insect resistance or tolerance; diseaseresistance or tolerance (viral, bacterial, fungal, nematode); stresstolerance and/or resistance, as exemplified by resistance or toleranceto drought, heat, chilling, freezing, excessive moisture, salt stressand oxidative stress; increased yields; food content and makeup;physical appearance; male sterility; drydown; standability; prolificacy;starch quantity and quality; oil quantity and quality; protein qualityand quantity; amino acid composition; and the like. Various U.S. Patentsdescribe ORFs that may be used to confer these traits, such as U.S. Pat.Nos. 5,550,318, 6,023,013 and 6,040,497, each incorporated herein byreference.

[0186] In certain embodiments of the invention, transformation of arecipient cell may be carried out with more than one exogenous(selected) gene (i.e., ORF). As used herein, an “exogenous codingregion” or “selected coding region” is a coding region not normallyfound in the host genome in an identical context. By this, it is meantthat the coding region may be isolated from a different species thanthat of the host genome, or alternatively, isolated from the hostgenome, but is operably linked to one or more regulatory regions whichdiffer from those found in the unaltered, native gene. Two or moreexogenous coding regions also can be supplied in a single transformationevent using either distinct transgene-encoding LEE or CEE constructs, orusing a single construct incorporating two or more coding sequences.

[0187] K. Gene Vaccines

[0188] In certain embodiments, the at least one LEE or CEE comprise orexpress an antigen. The antigen may promote an immune response by ananimal transfected or inoculated with the LEE or CEE encoding anantigen, or the LEE or CEE encoded antigen. Thus, the LEE or CEE maycomprise a vaccine or “gene vaccine” useful for immunization protocols.In this embodiment, ORFs encoding antigens for pathogenic (e.g., HIV,SIV, mycoplasma), parasitic or allergenic organisms are preferred.Additionally, ORFs for putative antigens or allergens may be assayed forimmunoreactions in one or more eukaryotic organisms.

[0189] The course of the immunization may be followed by assays forantibodies for the supernatant antigens. The assays may be performed bylabeling with conventional labels, such as radionuclides, enzymes,fluorescents, and the like. These techniques are well known and may befound in a wide variety of patents, such as U.S. Pat. Nos. 3,791,932;4,174,384 and 3,949,064, as illustrative of these types of assays. Otherimmune assays can be performed and assays of protection from challengewith the pathogen can be performed, following immunization.

[0190] 1. Immunomodulators

[0191] It is contemplated that immunomodulators can be included in thevaccine to augment the patient's response. Immunomodulators can beincluded as purified proteins or their expression engineered into thecells when cells are part of the composition. Genes encodingimmunomodulators can be included. The following sections list examplesof immunomodulators that are of interest.

[0192] a. Cytokines

[0193] Interleukins and cytokines, and vectors expressing interleukinsand cytokines are contemplated as possible vaccine components.Interleukins and cytokines, include but not limited to interleukin 1,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,IL-13, IL-14, IL-15, β-interferon, α-interferon, γ-interferon,angiostatin, thrombospondin, endostatin, METH-1, METH-2, GM-CSF, G-CSF,M-CSF, tumor necrosis factor, and combinations thereof.

[0194] a. Cofactors

[0195] Cofactors or genes that encode cofactors may be used in thevaccine. Examples of cofactors include, but are not limited to CD85,CD80, B7.1, B7.2, etc.

[0196] b. Chemokines

[0197] Chemokines or ORFs that code for chemokines also may be used asvaccine components. Chemokines generally act as chemoattractants torecruit immune effector cells to the site of chemokine expression. Itmay be advantageous to express a particular chemokine gene incombination with, for example, a cytokine gene, to enhance therecruitment of other immune system components to the site of treatment.Such chemokines include RANTES, MCAF, MIP1-alpha, MIP1-Beta, and IP-10.The skilled artisan will recognize that certain cytokines are also knownto have chemoattractant effects and could also be classified under theterm chemokines.

[0198] 2. Adjuvants

[0199] Immunization protocols have used adjuvants to stimulate responsesfor many years. Some adjuvants affect the way in which antigens arepresented. For example, the immune response is increased when proteinantigens are precipitated by alum. Emulsification of antigens alsoprolongs the duration of antigen presentation. Other adjuvants, forexample, certain organic molecules obtained from bacteria, act on thehost rather than on the antigen. An example is muramyl dipeptide(N-acetylmuramyl-L-alanyl-D-isoglutamine [MDP]), a bacterialpeptidoglycan. The effects of MDP, as with most adjuvants, are not fullyunderstood. MDP stimulates macrophages but also appears to stimulate Bcells directly. The effects of adjuvants, therefore, are notantigen-specific. If they are administered together with a purifiedantigen, however, they can be used to selectively promote the responseto the antigen.

[0200] Adjuvants have been used experimentally to promote a generalizedincrease in immunity against unknown antigens (e.g., U.S. Pat. No.4,877,611). This has been attempted particularly in the treatment ofcancer. For many cancers, there is compelling evidence that the immunesystem participates in host defense against the tumor cells, but only afraction of the likely total number of tumor-specific antigens arebelieved to have been identified to date. However, using the presentinvention, the inclusion of a suitable adjuvant into the membrane of anirradiated tumor cell will likely increase the anti-tumor responseirrespective of the molecular identification of the prominent antigens.This is a particularly important and time-saving feature of theinvention.

[0201] Those of skill in the art will know the different kinds ofadjuvants that can be conjugated to cellular vaccines in accordance withthis invention and these include alkyl lysophosphilipids (ALP); BCG; andbiotin (including biotinylated derivatives) among others. Certainadjuvants particularly contemplated for use are the teichoic acids fromGram −ve cells. These include the lipoteichoic acids (LTA), ribitolteichoic acids (RTA) and glycerol teichoic acid (GTA). Active forms oftheir synthetic counterparts may also be employed in connection with theinvention (Takada et al., 1995a).

[0202] Hemocyanins and hemoerythrins may also be used in the invention.The use of hemocyanin from keyhole limpet (KLH) is particularlypreferred, although other molluscan and arthropod hemocyanins andhemoerythrins may be employed.

[0203] Various polysaccharide adjuvants may also be used. For example,Yin et al., (1989) describe the use of various pneumococcalpolysaccharide adjuvants on the antibody responses of mice. The dosesthat produce optimal responses, or that otherwise do not producesuppression, as indicated in Yin et al., (1989) should be employed.Polyamine varieties of polysaccharides are particularly preferred, suchas chitin and chitosan, including deacetylated chitin.

[0204] A further preferred group of adjuvants are the muramyl dipeptide(MDP, N-acetylmuramyl-L-alanyl-D-isoglutaniine) group of bacterialpeptidoglycans. Derivatives of muramyl dipeptide, such as the amino acidderivative threonyl-MDP, and the fatty acid derivative MTPPE, are alsocontemplated.

[0205] U.S. Pat. No. 4,950,645 describes a lipophilicdisaccharide-tripeptide derivative of muramyl dipeptide which isproposed for use in artificial liposomes formed from phosphatidylcholine and phosphatidyl glycerol. It is said to be effective inactivating human monocytes and destroying tumor cells, but is non-toxicin generally high doses. The compounds of U.S. Pat. No. 4,950,645 andPCT Patent Application WO 91/16347, which have not previously beensuggested for use with cellular carriers, are now proposed for use inthe present invention.

[0206] A preferred adjuvant in the present invention is BCG. BCG(bacillus Calmette-Guerin, an attenuated strain of Mycobacterium) andBCG-cell wall skeleton (CWS) may also be used as adjuvants in theinvention, with or without trehalose dimycolate. Trehalose dimycolatemay be used itself. Azuma et al., (1988) show that trehalose dimycolateadministration correlates with augmented resistance to influenza virusinfection in mice. Trehalose dimycolate may be prepared as described inU.S. Pat. No. 4,579,945.

[0207] BCG is an important clinical tool because of itsimmunostimulatory properties. BCG acts to stimulate thereticulo-endothelial system, activates natural killer cells andincreases proliferation of hematopoietic stem cells. Cell wall extractsof BCG have proven to have excellent immune adjuvant activity. Recentlydeveloped molecular genetic tools and methods for mycobacteria haveprovided the means to introduce foreign genes into BCG (Jacobs et al.,1987; Snapper et al., 1988; Husson et al., 1990; Martin et al., 1990).Live BCG is an effective and safe vaccine used worldwide to preventtuberculosis. BCG and other mycobacteria are highly effective adjuvants,and the immune response to mycobacteria has been studied extensively.With nearly 2 billion immunizations, BCG has a long record of safe usein man (Luelmo, 1982; Lotte et al., 1984). It is one of the few vaccinesthat can be given at birth, it engenders long-lived immune responseswith only a single dose, and there is a worldwide distribution networkwith experience in BCG vaccination. An exemplary BCG vaccine is sold asTICEO BCG (Organon Inc., West Orange, N.J.).

[0208] In a typical practice of the present invention, cells ofMycobacterium bovis-BCG are grown and harvested by methods known in theart. For example, they may be grown as a surface pellicle on a Sautonmedium or in a fermentation vessel containing the dispersed culture in aDubos medium (Dubos et al., 1947; Rosenthal, 1937). All the cultures areharvested after 14 days incubation at about 37° C. Cells grown as apellicle are harvested by using a platinum loop whereas those from thefermenter are harvested by centrifugation or tangential-flow filtration.The harvested cells are re-suspended in an aqueous sterile buffermedium. A typical suspension contains from about 2×10¹⁰ cells/ml toabout 2×10¹² cells/ml. To this bacterial suspension, a sterile solutioncontaining a selected enzyme which will degrade the BCG cell coveringmaterial is added. The resultant suspension is agitated such as bystirring to ensure maximal dispersal of the BCG organisms. Thereafter, amore concentrated cell suspension is prepared and the enzyme in theconcentrate removed, typically by washing with an aqueous buffer,employing known techniques such as tangential-flow filtration. Theenzyme-free cells are adjusted to an optimal immunological concentrationwith a cryoprotectant solution, after which they are filled into vials,ampoules, etc., and lyophilized, yielding BCG vaccine, which uponreconstitution with water is ready for immunization.

[0209] Amphipathic and surface active agents, e.g., saponin andderivatives such as QS21 (Cambridge Biotech), form yet another group ofpreferred adjuvants for use with the immunogens of the presentinvention. Nonionic block copolymer surfactants (Rabinovich et al.,1994; Hunter et al., 1991) may also be employed. Oligonucleotides, asdescribed by Yamamoto et al., (1988) are another useful group ofadjuvants. Quil A and lentinen complete the currently preferred list ofadjuvants. Although each of the agents, and the endotoxins describedbelow, are well-known as adjuvants, these compounds have not beenpreviously incorporated into the membrane of a target cell, as shownherein.

[0210] One group of adjuvants preferred for use in the invention are thedetoxified endotoxins, such as the refined detoxified endotoxin of U.S.Pat. No. 4,866,034. These refined detoxified endotoxins are effective inproducing adjuvant responses in mammals.

[0211] The detoxified endotoxins may be combined with other adjuvants toprepare multi-adjuvant-incorporated cells. Combination of detoxifiedendotoxins with trehalose dimycolate is contemplated, as described inU.S. Pat. No. 4,435,386. Combinations of detoxified endotoxins withtrehalose dimycolate and endotoxic glycolipids is also contemplated(U.S. Pat. No. 4,505,899), as is combination of detoxified endotoxinswith cell wall skeleton (CWS) or CWS and trehalose dimycolate, asdescribed in U.S. Pat. Nos. 4,436,727, 4,436,728 and 4,505,900.Combinations of just CWS and trehalose dimycolate, without detoxifiedendotoxins, is also envisioned to be useful, as described in U.S. Pat.No. 4,520,019.

[0212] One group of adjuvants particularly preferred for use in thepresent invention are those that can be encoded by DNA or RNA. It iscontemplated that such adjuvants may be encoded in a LEE or CEE vectorencoding the antigen, or as separate LEE or CEE vectors, or tradtionalplasmids or other constructs. These nucleic acids encoding the adjuvantscan be delivered directly, such as for example with lipids or liposomes.A LEE or CEE encoding antigens might also be formulated withproteinaceous adjuvants in a lipid or liposome.

[0213] Various adjuvants, even those that are not commonly used inhumans, may still be employed in animals, where, for example, onedesires to raise antibodies or to subsequently obtain activated T cells.The toxicity or other adverse effects that may result from either theadjuvant or the cells, e.g., as may occur using non-irradiated tumorcells, is irrelevant in such circumstances.

[0214] Vaccines may be conventionally administered parenterally, byinjection, for example, either subcutaneously, intradermally orintramuscularly. In many instances, it will be desirable to havemultiple administrations of the vaccine, usually not exceeding sixvaccinations, more usually not exceeding four vaccinations andpreferably one or more, usually at least about three vaccinations. Thevaccinations will normally be at from two to twelve week intervals, moreusually from three to five week intervals. Periodic boosters atintervals of 1-5 years, usually three years, will be desirable tomaintain protective levels of the antibodies.

[0215] L. Immunological Reagents

[0216] In certain aspects of the invention, one or more antibodies maybe produced to the expressed ORF. These antibodies may be used invarious diagnostic or therapeutic applications, described herein below.

[0217] As used herein, the term “antibody” is intended to refer broadlyto any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE.Generally, IgG and/or IgM are preferred because they are the most commonantibodies in the physiological situation and because they are mosteasily made in a laboratory setting.

[0218] The term “antibody” is used to refer to any antibody-likemolecule that has an antigen binding region, and includes antibodyfragments such as Fab′, Fab, F(ab′)₂, single domain antibodies (DABs),Fv, scFv (single chain Fv), and the like. The techniques for preparingand using various antibody-based constructs and fragments are well knownin the art. Means for preparing and characterizing antibodies are alsowell known in the art (See, e.g., Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988; incorporated herein by reference).

[0219] Monoclonal antibodies (MAbs) are recognized to have certainadvantages, e.g., reproducibility and large-scale production, and theiruse is generally preferred. The invention thus provides monoclonalantibodies of the human, murine, monkey, rat, hamster, rabbit and evenchicken origin. Due to the ease of preparation and ready availability ofreagents, murine monoclonal antibodies will often be preferred.

[0220] However, “humanized” antibodies are also contemplated, as arechimeric antibodies from mouse, rat, or other species, bearing humanconstant and/or variable region domains, bispecific antibodies,recombinant and engineered antibodies and fragments thereof. Methods forthe development of antibodies that are “custom-tailored” to thepatient's dental disease are likewise known and such custom-tailoredantibodies are also contemplated.

[0221] The methods for generating monoclonal antibodies (MAbs) generallybegin along the same lines as those for preparing polyclonal antibodies.Briefly, a polyclonal antibody is prepared by immunizing an animal witha LEE or CEE composition in accordance with the present invention andcollecting antisera from that immunized animal.

[0222] A wide range of animal species can be used for the production ofantisera. Typically the animal used for production of antisera is arabbit, a mouse, a rat, a hamster, a guinea pig or a goat. The choice ofanimal may be decided upon the ease of manipulation, costs or thedesired amount of sera, as would be known to one of skill in the art.

[0223] As is also well known in the art, the immunogenicity of aparticular immunogen composition can be enhanced by the use ofnon-specific stimulators of the immune response, known as adjuvants.Suitable adjuvants include all acceptable immunostimulatory compounds,such as cytokines, chemokines, cofactors, toxins, plasmodia, syntheticcompositions or LEEs or CEEs encoding such adjuvants.

[0224] Adjuvants that may be used include IL-1, IL-2, IL-4, IL-7, IL-12,γ-interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds, such asthur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A(MPL). RIBI, which contains three components extracted from bacteria,MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2%squalene/Tween 80 emulsion is also contemplated. MHC antigens may evenbe used. Exemplary, often preferred adjuvants include complete Freund'sadjuvant (a non-specific stimulator of the immune response containingkilled Mycobacterium tuberculosis), incomplete Freund's adjuvants andaluminum hydroxide adjuvant.

[0225] In addition to adjuvants, it may be desirable to coadministerbiologic response modifiers (BRM), which have been shown to upregulate Tcell immunity or downregulate suppressor cell activity. Such BRMsinclude, but are not limited to, Cimetidine (CIM; 1200 mg/d)(Smith/Kline, PA); low-dose Cyclophosphamide (CYP; 300 mg/m²)(Johnson/Mead, NJ), cytokines such as γ-interferon, IL-2, or IL-12 orgenes encoding proteins involved in immune helper functions, such asB-7.

[0226] The amount of immunogen composition used in the production ofpolyclonal antibodies varies upon the nature of the immunogen as well asthe animal used for immunization. A variety of routes can be used toadminister the immunogen including but not limited to subcutaneous,intramuscular, intradermal, intraepidermal, intravenous andintraperitoneal. The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization.

[0227] A second, booster dose (e.g., provided in an injection), may alsobe given. The process of boosting and titering is repeated until asuitable titer is achieved. When a desired level of immunogenicity isobtained, the immunized animal can be bled and the serum isolated andstored, and/or the animal can be used to generate MAbs.

[0228] For production of rabbit polyclonal antibodies, the animal can bebled through an ear vein or alternatively by cardiac puncture. Theremoved blood is allowed to coagulate and then centrifuged to separateserum components from whole cells and blood clots. The serum may be usedas is for various applications or else the desired antibody fraction maybe purified by well-known methods, such as affinity chromatography usinganother antibody, a peptide bound to a solid matrix, or by using, e.g.,protein A or protein G chromatography.

[0229] MAbs may be readily prepared through use of well-knowntechniques, such as those exemplified in U.S. Pat. No. 4,196,265,incorporated herein by reference. Typically, this technique involvesimmunizing a suitable animal with a selected immunogen composition,e.g., a purified or partially purified protein, polypeptide, peptide ordomain, be it a wild-type or mutant composition. The immunizingcomposition is administered in a manner effective to stimulate antibodyproducing cells.

[0230] The methods for generating monoclonal antibodies (MAbs) generallybegin along the same lines as those for preparing polyclonal antibodies.Rodents such as mice and rats are preferred animals, however, the use ofrabbit, sheep or frog cells is also possible. The use of rats mayprovide certain advantages (Goding, 1986, pp. 60-61), but mice arepreferred, with the BALB/c mouse being most preferred as this is mostroutinely used and generally gives a higher percentage of stablefusions.

[0231] The animals are injected with antigen, generally as describedabove. The antigen may be mixed with adjuvant, such as Freund's completeor incomplete adjuvant. Booster administrations with the same antigen orDNA encoding the antigen would occur at approximately two-weekintervals.

[0232] Following immunization, somatic cells with the potential forproducing antibodies, specifically B lymphocytes (B cells), are selectedfor use in the MAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible.

[0233] Often, a panel of animals will have been immunized and the spleenof an animal with the highest antibody titer will be removed and thespleen lymphocytes obtained by homogenizing the spleen with a syringe.Typically, a spleen from an immunized mouse contains approximately 5×10⁷to 2×10⁸ lymphocytes.

[0234] The antibody-producing B lymphocytes from the immunized animalare then fused with cells of an immortal myeloma cell, generally one ofthe same species as the animal that was immunized. Myeloma cell linessuited for use in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

[0235] Any one of a number of myeloma cells may be used, as are known tothose of skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83,1984). cites). For example, where the immunized animal is a mouse, onemay use P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U,MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may useR210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2,LICR-LON-HMy2 and UC729-6 are all useful in connection with human cellfusions.

[0236] One preferred murine myeloma cell is the NS-1 myeloma cell line(also termed P3-NS-1-Ag4-1), which is readily available from the NIGMSHuman Genetic Mutant Cell Repository by requesting cell line repositorynumber GM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cellline.

[0237] Methods for generating hybrids of antibody-producing spleen orlymph node cells and myeloma cells usually comprise mixing somatic cellswith myeloma cells in a 2:1 proportion, though the proportion may varyfrom about 20:1 to about 1:1, respectively, in the presence of an agentor agents (chemical or electrical) that promote the fusion of cellmembranes. Fusion methods using Sendai virus have been described byKohler and Milstein (1975; 1976), and those using polyethylene glycol(PEG), such as 37% (v/v) PEG, by Gefter et al., (1977). The use ofelectrically induced fusion methods is also appropriate (Goding pp.71-74, 1986).

[0238] Fusion procedures usually produce viable hybrids at lowfrequencies, about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose aproblem, as the viable, fused hybrids are differentiated from theparental, unfused cells (particularly the unfused myeloma cells thatwould normally continue to divide indefinitely) by culturing in aselective medium. The selective medium is generally one that contains anagent that blocks the de novo synthesis of nucleotides in the tissueculture media. Exemplary and preferred agents are aminopterin,methotrexate, and azaserine. Aminopterin and methotrexate block de novosynthesis of both purines and pyrimidines, whereas azaserine blocks onlypurine synthesis. Where aminopterin or methotrexate is used, the mediais supplemented with hypoxanthine and thymidine as a source ofnucleotides (HAT medium). Where azaserine is used, the media issupplemented with hypoxanthine.

[0239] The preferred selection medium is HAT. Only cells capable ofoperating nucleotide salvage pathways are able to survive in HAT medium.The myeloma cells are defective in key enzymes of the salvage pathway,e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannotsurvive. The B cells can operate this pathway, but they have a limitedlife span in culture and generally die within about two weeks.Therefore, the only cells that can survive in the selective media arethose hybrids formed from myeloma and B cells.

[0240] This culturing provides a population of hybridomas from whichspecific hybridomas are selected. Typically, selection of hybridomas isperformed by culturing the cells by single-clone dilution in microtiterplates, followed by testing the individual clonal supernatants (afterabout two to three weeks) for the desired reactivity. The assay shouldbe sensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

[0241] The selected hybridomas would then be serially diluted and clonedinto individual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide MAbs. The cell lines may be exploitedfor MAb production in two basic ways. First, a sample of the hybridomacan be injected (often into the peritoneal cavity) into ahistocompatible animal of the type that was used to provide the somaticand myeloma cells for the original fusion (e.g., a syngeneic mouse).Optionally, the animals are primed with a hydrocarbon, especially oilssuch as pristane (tetramethylpentadecane) prior to injection. Theinjected animal develops tumors secreting the specific monoclonalantibody produced by the fused cell hybrid. The body fluids of theanimal, such as serum or ascites fluid, can then be tapped to provideMAbs in high concentration. Second, the individual cell lines could becultured in vitro, where the MAbs are naturally secreted into theculture medium from which they can be readily obtained in highconcentrations.

[0242] MAbs produced by either means may be further purified, ifdesired, using filtration, centrifugation and various chromatographicmethods such as HPLC or affinity chromatography. Fragments of themonoclonal antibodies of the invention can be obtained from themonoclonal antibodies so produced by methods which include digestionwith enzymes, such as pepsin or papain, and/or by cleavage of disulfidebonds by chemical reduction. Alternatively, monoclonal antibodyfragments encompassed by the present invention can be synthesized usingan automated peptide synthesizer.

[0243] It is also contemplated that a molecular cloning approach may beused to generate monoclonals. In one embodiment, combinatorialimmunoglobulin phagemid libraries are prepared from RNA isolated fromthe spleen of the immunized animal, and phagemids expressing appropriateantibodies are selected by panning using cells expressing the antigenand control cells. The advantages of this approach over conventionalhybridoma techniques are that approximately 10⁴ times as many antibodiescan be produced and screened in a single round, and that newspecificities are generated by H and L chain combination which furtherincreases the chance of finding appropriate antibodies. In anotherexample, LEEs or CEEs can be used to produce antigens in vitro with acell free system. These can be used as targets for scanning single chainantibody libraries. This would enable many different antibodies to beidentified very quickly without the use of animals.

[0244] Alternatively, monoclonal antibody fragments encompassed by thepresent invention can be synthesized using an automated peptidesynthesizer, or by expression of full-length gene or of gene fragmentsin E. coli.

[0245] 1. Antibody Conjugates

[0246] The present invention further provides antibodies to ORFtranscribed messages and translated proteins, polypeptides and peptides,generally of the monoclonal type, that are linked to at least one agentto form an antibody conjugate. In order to increase the efficacy ofantibody molecules as diagnostic or therapeutic agents, it isconventional to link or covalently bind or complex at least one desiredmolecule or moiety. Such a molecule or moiety may be, but is not limitedto, at least one effector or reporter molecule. Effector moleculescomprise molecules having a desired activity, e.g., cytotoxic activity.Non-limiting examples of effector molecules which have been attached toantibodies include toxins, anti-tumor agents, therapeutic enzymes,radio-labeled nucleotides, antiviral agents, chelating agents,cytokines, growth factors, and oligo- or poly-nucleotides. By contrast,a reporter molecule is defined as any moiety which may be detected usingan assay. Non-limiting examples of reporter molecules which have beenconjugated to antibodies include enzymes, radiolabels, haptens,fluorescent labels, phosphorescent molecules, chemiluminescentmolecules, chromophores, luminescent molecules, photoaffinity molecules,colored particles or ligands, such as biotin.

[0247] Any antibody of sufficient selectivity, specificity or affinitymay be employed as the basis for an antibody conjugate. Such propertiesmay be evaluated using conventional immunological screening methodologyknown to those of skill in the art. Sites for binding to biologicalactive molecules in the antibody molecule, in addition to the canonicalantigen binding sites, include sites that reside in the variable domainthat can bind pathogens, B-cell superantigens, the T cell co-receptorCD4 and the HIV-1 envelope (Sassoetal., 1989; Shorkietal., 1991;Silvermann et al., 1995; Cleary et al., 1994; Lenert et al., 1990;Berberian et al., 1993; Kreier et al., 1991). In addition, the variabledomain is involved in antibody self-binding (Kang et al., 1988), andcontains epitopes (idiotopes) recognized by anti-antibodies (Kohler etal., 1989).

[0248] Certain examples of antibody conjugates are those conjugates inwhich the antibody is linked to a detectable label. “Detectable labels”are compounds and/or elements that can be detected due to their specificfunctional properties, and/or chemical characteristics, the use of whichallows the antibody to which they are attached to be detected, and/orfurther quantified if desired. Another such example is the formation ofa conjugate comprising an antibody linked to a cytotoxic oranti-cellular agent, and may be termed “immunotoxins”.

[0249] Antibody conjugates are generally preferred for use as diagnosticagents. Antibody diagnostics generally fall within two classes, thosefor use in in vitro diagnostics, such as in a variety of immunoassays,and/or those for use in vivo diagnostic protocols, generally known as“antibody-directed imaging”.

[0250] Many appropriate imaging agents are known in the art, as aremethods for their attachment to antibodies (see, for e.g., U.S. Pat.Nos. 5,021,236; 4,938,948; and 4,472,509, each incorporated herein byreference). The imaging moieties used can be paramagnetic ions;radioactive isotopes; fluorochromes; NMR-detectable substances; X-rayimaging.

[0251] In the case of paramagnetic ions, one might mention by way ofexample ions such as chromium (III), manganese (II), iron (III), iron(II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium(III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and/or erbium (III), with gadoliniumbeing particularly preferred. Ions useful in other contexts, such asX-ray imaging, include but are not limited to lanthanum (III), gold(III), lead (II), and especially bismuth (III).

[0252] In the case of radioactive isotopes for therapeutic and/ordiagnostic application, one might mention astatine²¹¹, ¹⁴carbon,⁵¹chromium, ³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt, copper⁶⁷, ¹⁵²Eu, gallium⁶⁷,³hydrogen, iodine¹²³, iodine¹²⁵, iodine¹³¹, indium¹¹¹, ⁵⁹iron,³²phosphorus, rhenium¹⁸⁶, rhenium¹⁸⁸, ⁷⁵selenium, ³⁵sulphur,technicium^(99m) and/or yttrium⁹⁰. ¹²⁵I is often being preferred for usein certain embodiments, and technicium^(99m) and/or indium¹¹¹ are alsooften preferred due to their low energy and suitability for long rangedetection. Radioactively labeled monoclonal antibodies of the presentinvention may be produced according to well-known methods in the art.For instance, monoclonal antibodies can be iodinated by contact withsodium and/or potassium iodide and a chemical oxidizing agent such assodium hypochlorite, or an enzymatic oxidizing agent, such aslactoperoxidase. Monoclonal antibodies according to the invention may belabeled with technetium^(99m) by ligand exchange process, for example,by reducing pertechnate with stannous solution, chelating the reducedtechnetium onto a Sephadex column and applying the antibody to thiscolumn. Alternatively, direct labeling techniques may be used, e.g., byincubating pertechnate, a reducing agent such as SNCl₂, a buffersolution such as sodium-potassium phthalate solution, and the antibody.Intermediary functional groups which are often used to bindradioisotopes which exist as metallic ions to antibody arediethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetraceticacid (EDTA).

[0253] Among the fluorescent labels contemplated for use as conjugatesinclude Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665,BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3,Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488,Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green,Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine,and/or Texas Red.

[0254] Another type of antibody conjugates contemplated in the presentinvention are those intended primarily for use in vitro, where theantibody is linked to a secondary binding ligand and/or to an enzyme (anenzyme tag) that will generate a colored product upon contact with achromogenic substrate. Examples of suitable enzymes include urease,alkaline phosphatase, (horseradish) hydrogen peroxidase or glucoseoxidase. Preferred secondary binding ligands are biotin and/or avidinand streptavidin compounds. The use of such labels is well known tothose of skill in the art and are described, for example, in U.S. Pat.Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149and 4,366,241; each incorporated herein by reference.

[0255] Yet another known method of site-specific attachment of moleculesto antibodies comprises the reaction of antibodies with hapten-basedaffinity labels. Essentially, hapten-based affinity labels react withamino acids in the antigen binding site, thereby destroying this siteand blocking specific antigen reaction. However, this may not beadvantageous since it results in loss of antigen binding by the antibodyconjugate.

[0256] Molecules containing azido groups may also be used to formcovalent bonds to proteins through reactive nitrene intermediates thatare generated by low intensity ultraviolet light (Potter & Haley, 1983).In particular, 2- and 8-azido analogues of purine nucleotides have beenused as site-directed photoprobes to identify nucleotide bindingproteins in crude cell extracts (Owens & Haley, 1987; Atherton et al.,1985). The 2- and 8-azido nucleotides have also been used to mapnucleotide binding domains of purified proteins (Khatoon et al., 1989;King et al., 1989; and Dholakia et al., 1989) and may be used asantibody binding agents.

[0257] Several methods are known in the art for the attachment orconjugation of an antibody to its conjugate moiety. Some attachmentmethods involve the use of a metal chelate complex employing, forexample, an organic chelating agent such a diethylenetriaminepentaaceticacid anhydride (DTPA); ethylenetriaminetetraacetic acid;N-chloro-p-toluenesulfonamide; and/ortetrachloro-3a-6a-diphenylglycouril-3 attached to the antibody (U.S.Pat. Nos. 4,472,509 and 4,938,948, each incorporated herein byreference). Monoclonal antibodies may also be reacted with an enzyme inthe presence of a coupling agent such as glutaraldehyde or periodate.Conjugates with fluorescein markers are prepared in the presence ofthese coupling agents or by reaction with an isothiocyanate. In U.S.Pat. No. 4,938,948, imaging of breast tumors is achieved usingmonoclonal antibodies and the detectable imaging moieties are bound tothe antibody using linkers such as methyl-p-hydroxybenzimidate orN-succinimidyl-3-(4-hydroxyphenyl)propionate.

[0258] In other embodiments, derivatization of immunoglobulins byselectively introducing sulfhydryl groups in the Fc region of animmunoglobulin, using reaction conditions that do not alter the antibodycombining site are contemplated. Antibody conjugates produced accordingto this methodology are disclosed to exhibit improved longevity,specificity and sensitivity (U.S. Pat. No. 5,196,066, incorporatedherein by reference). Site-specific attachment of effector or reportermolecules, wherein the reporter or effector molecule is conjugated to acarbohydrate residue in the Fe region have also been disclosed in theliterature (O'Shannessy et al., 1987). This approach has been reportedto produce diagnostically and therapeutically promising antibodies whichare currently in clinical evaluation.

[0259] 2. Immunodetection Methods

[0260] In still further embodiments, the present invention concernsimmunodetection methods for binding, purifying, removing, quantifyingand/or otherwise generally detecting biological components such as ORFexpressed message(s), protein(s), polypeptide(s) or peptide(s). Someimmunodetection methods include enzyme linked immunosorbent assay(ELISA), radioimmunoassay (RIA), immunoradiometric assay,fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, andWestern blot to mention a few. The steps of various usefulimmunodetection methods have been described in the scientificliterature, such as, e.g., Doolittle M H and Ben-Zeev O, 1999; Gulbis Band Galand P, 1993; De Jager R et al., 1993; and Nakamura et al., 1987,each incorporated herein by reference.

[0261] In general, the immunobinding methods include obtaining a samplesuspected of containing ORF expressed message and/or protein,polypeptide and/or peptide, and contacting the sample with a firstanti-ORF message and/or anti-ORF translated product antibody inaccordance with the present invention, as the case may be, underconditions effective to allow the formation of immunocomplexes.

[0262] These methods include methods for purifying an ORF message,protein, polypeptide and/or peptide from organelle, cell, tissue ororganism's samples. In these instances, the antibody removes theantigenic ORF message, protein, polypeptide and/or peptide componentfrom a sample. The antibody will preferably be linked to a solidsupport, such as in the form of a column matrix, and the samplesuspected of containing the ORF message, protein, polypeptide and/orpeptide antigenic component will be applied to the immobilized antibody.The unwanted components will be washed from the column, leaving theantigen immunocomplexed to the immobilized antibody to be eluted.

[0263] The immunobinding methods also include methods for detecting andquantifying the amount of an antigen component in a sample and thedetection and quantification of any immune complexes formed during thebinding process. Here, one would obtain a sample suspected of containingan antigen, and contact the sample with an antibody against the ORFproduced antigen, and then detect and quantify the amount of immunecomplexes formed under the specific conditions.

[0264] In terms of antigen detection, the biological sample analyzed maybe any sample that is suspected of containing an antigen, such as, forexample, a tissue section or specimen, a homogenized tissue extract, acell, an organelle, separated and/or purified forms of any of the aboveantigen-containing compositions, or even any biological fluid that comesinto contact with the cell or tissue, including blood and/or serum,although tissue samples or extracts are preferred.

[0265] Contacting the chosen biological sample with the antibody undereffective conditions and for a period of time sufficient to allow theformation of immune complexes (primary immune complexes) is generally amatter of simply adding the antibody composition to the sample andincubating the mixture for a period of time long enough for theantibodies to form immune complexes with, i.e., to bind to, any ORFantigens present. After this time, the sample-antibody composition, suchas a tissue section, ELISA plate, dot blot or western blot, willgenerally be washed to remove any non-specifically bound antibodyspecies, allowing only those antibodies specifically bound within theprimary immune complexes to be detected.

[0266] In general, the detection of immunocomplex formation is wellknown in the art and may be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any of those radioactive, fluorescent,biological and enzymatic tags. U.S. Patents concerning the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated hereinby reference. Of course, one may find additional advantages through theuse of a secondary binding ligand such as a second antibody and/or abiotin/avidin ligand binding arrangement, as is known in the art.

[0267] The ORF antigen antibody employed in the detection may itself belinked to a detectable label, wherein one would then simply detect thislabel, thereby allowing the amount of the primary immune complexes inthe composition to be determined. Alternatively, the first antibody thatbecomes bound within the primary immune complexes may be detected bymeans of a second binding ligand that has binding affinity for theantibody. In these cases, the second binding ligand may be linked to adetectable label. The second binding ligand is itself often an antibody,which may thus be termed a “secondary” antibody. The primary immunecomplexes are contacted with the labeled, secondary binding ligand, orantibody, under effective conditions and for a period of time sufficientto allow the formation of secondary immune complexes. The secondaryimmune complexes are then generally washed to remove anynon-specifically bound labeled secondary antibodies or ligands, and theremaining label in the secondary immune complexes is then detected.

[0268] Further methods include the detection of primary immune complexesby a two step approach. A second binding ligand, such as an antibody,that has binding affinity for the antibody is used to form secondaryimmune complexes, as described above. After washing, the secondaryimmune complexes are contacted with a third binding ligand or antibodythat has binding affinity for the second antibody, again under effectiveconditions and for a period of time sufficient to allow the formation ofimmune complexes (tertiary immune complexes). The third ligand orantibody is linked to a detectable label, allowing detection of thetertiary immune complexes thus formed. This system may provide forsignal amplification if this is desired.

[0269] One method of immunodetection designed by Charles Cantor uses twodifferent antibodies. A first step biotinylated, monoclonal orpolyclonal antibody is used to detect the target antigen(s), and asecond step antibody is then used to detect the biotin attached to thecomplexed biotin. In that method the sample to be tested is firstincubated in a solution containing the first step antibody. If thetarget antigen is present, some of the antibody binds to the antigen toform a biotinylated antibody/antigen complex. The antibody/antigencomplex is then amplified by incubation in successive solutions ofstreptavidin (or avidin), biotinylated DNA, and/or complementarybiotinylated DNA, with each step adding additional biotin sites to theantibody/antigen complex. The amplification steps are repeated until asuitable level of amplification is achieved, at which point the sampleis incubated in a solution containing the second step antibody againstbiotin. This second step antibody is labeled, as for example with anenzyme that can be used to detect the presence of the antibody/antigencomplex by histoenzymology using a chromogen substrate. With suitableamplification, a conjugate can be produced which is macroscopicallyvisible.

[0270] Another known method of immunodetection takes advantage of theimmuno-PCR (Polymerase Chain Reaction) methodology. The PCR method issimilar to the Cantor method up to the incubation with biotinylated DNA,however, instead of using multiple rounds of streptavidin andbiotinylated DNA incubation, the DNA/biotin/streptavidin/antibodycomplex is washed out with a low pH or high salt buffer that releasesthe antibody. The resulting wash solution is then used to carry out aPCR reaction with suitable primers with appropriate controls. At leastin theory, the enormous amplification capability and specificity of PCRcan be utilized to detect a single antigen molecule.

[0271] The immunodetection methods of the present invention have evidentutility in the diagnosis and prognosis of conditions such as variousdiseases wherein a specific ORF is expressed, such as an viral ORF of aviral infected cell, tissue or organism; a cancer specific gene product,etc. Here, a biological and/or clinical sample suspected of containing aspecific disease associated ORF expression product is used. However,these embodiments also have applications to non-clinical samples, suchas in the titering of antigen or antibody samples, for example in theselection of hybridomas.

[0272] In the clinical diagnosis and/or monitoring of patients withvarious forms a disease, such as, for example, cancer, the detection ofa cancer specific ORF gene product, and/or an alteration in the levelsof a cancer specific gene product, in comparison to the levels in acorresponding biological sample from a normal subject is indicative of apatient with cancer. However, as is known to those of skill in the art,such a clinical diagnosis would not necessarily be made on the basis ofthis method in isolation. Those of skill in the art are very familiarwith differentiating between significant differences in types and/oramounts of biomarkers, which represent a positive identification, and/orlow level and/or background changes of biomarkers. Indeed, backgroundexpression levels are often used to form a “cut-off” above whichincreased detection will be scored as significant and/or positive. Ofcourse, the antibodies of the present invention in any immunodetectionor therapy known to one of ordinary skill in the art.

[0273] a. ELISAs

[0274] As detailed above, immunoassays, in their most simple and/ordirect sense, are binding assays. Certain preferred immunoassays are thevarious types of enzyme linked immunosorbent assays (ELISAs) and/orradioimmunoassays (RIA) known in the art. Immunohistochemical detectionusing tissue sections is also particularly useful. However, it will bereadily appreciated that detection is not limited to such techniques,and/or western blotting, dot blotting, FACS analyses, and/or the likemay also be used.

[0275] In one exemplary ELISA, the anti-ORF message and/or anti-ORFtranslated product antibodies of the invention are immobilized onto aselected surface exhibiting protein affinity, such as a well in apolystyrene microtiter plate. Then, a test composition suspected ofcontaining the antigen, such as a clinical sample, is added to thewells. After binding and/or washing to remove non-specifically boundimmune complexes, the bound antigen may be detected. Detection isgenerally achieved by the addition of another anti-ORF message and/oranti-ORF translated product antibody that is linked to a detectablelabel. This type of ELISA is a simple “sandwich ELISA”. Detection mayalso be achieved by the addition of a second anti-ORF message and/oranti-ORF translated product antibody, followed by the addition of athird antibody that has binding affinity for the second antibody, withthe third antibody being linked to a detectable label.

[0276] In another exemplary ELISA, the samples suspected of containingthe antigen are immobilized onto the well surface and/or then contactedwith the anti-ORF message and/or anti-ORF translated product antibodiesof the invention. After binding and/or washing to removenon-specifically bound immune complexes, the bound anti-ORF messageand/or anti-ORF translated product antibodies are detected. Where theinitial anti-ORF message and/or anti-ORF translated product antibodiesare linked to a detectable label, the immune complexes may be detecteddirectly. Again, the immune complexes may be detected using a secondantibody that has binding affinity for the first anti-ORF message and/oranti-ORF translated product antibody, with the second antibody beinglinked to a detectable label.

[0277] Another ELISA in which the antigens are immobilized, involves theuse of antibody competition in the detection. In this ELISA, labeledantibodies against an antigen are added to the wells, allowed to bind,and/or detected by means of their label. The amount of an antigen in anunknown sample is then determined by mixing the sample with the labeledantibodies against the antigen during incubation with coated wells. Thepresence of an antigen in the sample acts to reduce the amount ofantibody against the antigen available for binding to the well and thusreduces the ultimate signal. This is also appropriate for detectingantibodies against an antigen in an unknown sample, where the unlabeledantibodies bind to the antigen-coated wells and also reduces the amountof antigen available to bind the labeled antibodies.

[0278] Irrespective of the format employed, ELISAs have certain featuresin common, such as coating, incubating and binding, washing to removenon-specifically bound species, and detecting the bound immunecomplexes. These are described below.

[0279] In coating a plate with either antigen or antibody, one willgenerally incubate the wells of the plate with a solution of the antigenor antibody, either overnight or for a specified period of hours. Thewells of the plate will then be washed to remove incompletely adsorbedmaterial. Any remaining available surfaces of the wells are then“coated” with a nonspecific protein that is antigenically neutral withregard to the test antisera. These include bovine serum albumin (BSA),casein or solutions of milk powder. The coating allows for blocking ofnonspecific adsorption sites on the immobilizing surface and thusreduces the background caused by nonspecific binding of antisera ontothe surface.

[0280] In ELISAs, it is probably more customary to use a secondary ortertiary detection means rather than a direct procedure. Thus, afterbinding of a protein or antibody to the well, coating with anon-reactive material to reduce background, and washing to removeunbound material, the immobilizing surface is contacted with thebiological sample to be tested under conditions effective to allowimmune complex (antigen/antibody) formation. Detection of the immunecomplex then requires a labeled secondary binding ligand or antibody,and a secondary binding ligand or antibody in conjunction with a labeledtertiary antibody or a third binding ligand.

[0281] “Under conditions effective to allow immune complex(antigen/antibody) formation” means that the conditions preferablyinclude diluting the antigens and/or antibodies with solutions such asBSA, bovine gamma globulin (BGG) or phosphate buffered saline(PBS)/Tween. These added agents also tend to assist in the reduction ofnonspecific background.

[0282] The “suitable” conditions also mean that the incubation is at atemperature or for a period of time sufficient to allow effectivebinding. Incubation steps are typically from about 1 to 2 to 4 hours orso, at temperatures preferably on the order of 25° C. to 27° C., or maybe overnight at about 4° C. or so.

[0283] Following all incubation steps in an ELISA, the contacted surfaceis washed so as to remove non-complexed material. A preferred washingprocedure includes washing with a solution such as PBS/Tween, or boratebuffer. Following the formation of specific immune complexes between thetest sample and the originally bound material, and subsequent washing,the occurrence of even minute amounts of immune complexes may bedetermined.

[0284] To provide a detecting means, the second or third antibody willhave an associated label to allow detection. Preferably, this will be anenzyme that will generate color development upon incubating with anappropriate chromogenic substrate. Thus, for example, one will desire tocontact or incubate the first and second immune complex with a urease,glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugatedantibody for a period of time and under conditions that favor thedevelopment of further immune complex formation (e.g., incubation for 2hours at room temperature in a PBS-containing solution such asPBS-Tween).

[0285] After incubation with the labeled antibody, and subsequent towashing to remove unbound material, the amount of label is quantified,e.g., by incubation with a chromogenic substrate such as urea, orbromocresol purple, or 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonicacid (ABTS), or H₂O₂, in the case of peroxidase as the enzyme label.Quantification is then achieved by measuring the degree of colorgenerated, e.g., using a visible spectra spectrophotometer.

[0286] b. Immunohistochemistry

[0287] The antibodies of the present invention may also be used inconjunction with both fresh-frozen and/or formalin-fixed,paraffin-embedded tissue blocks prepared for study byimmunohistochemistry (IHC). The method of preparing tissue blocks fromthese particulate specimens has been successfully used in previous IHCstudies of various prognostic factors, and/or is well known to those ofskill in the art (Brown et al., 1990; Abbondanzo et al., 1990; Allred etal., 1990).

[0288] Briefly, frozen-sections may be prepared by rehydrating 50 ng offrozen “pulverized” tissue at room temperature in phosphate bufferedsaline (PBS) in small plastic capsules; pelleting the particles bycentrifugation; resuspending them in a viscous embedding medium (OCT);inverting the capsule and/or pelleting again by centrifugation;snap-freezing in −70° C. isopentane; cutting the plastic capsule and/orremoving the frozen cylinder of tissue; securing the tissue cylinder ona cryostat microtome chuck; and/or cutting 25-50 serial sections.

[0289] Permanent-sections may be prepared by a similar method involvingrehydration of the 50 mg sample in a plastic microfuge tube; pelleting;resuspending in 10% formalin for 4 hours fixation; washing/pelleting;resuspending in warm 2.5% agar; pelleting; cooling in ice water toharden the agar; removing the tissue/agar block from the tube;infiltrating and/or embedding the block in paraffin; and/or cutting upto 50 serial permanent sections.

[0290] M. Assays of Gene Expression

[0291] Assays may be employed within the scope of the instant inventionfor determination of the relative efficiency of expression from a LEE orCEE. For example, assays may be used to determine the efficacy ofdeletion mutants of specific promoter regions in directing expression ofoperably linked genes. Similarly, one could produce random orsite-specific mutants of promoter regions and assay the efficacy of themutants in the expression of an operably linked gene. Alternatively,assays could be used to determine the function of a promoter region inenhancing gene expression when used in conjunction with variousdifferent regulatory elements, enhancers, and exogenous genes.

[0292] Gene expression may be determined by measuring the production ofRNA, protein or both, or a consequence of RNA protein, polypeptide orpeptide production. The gene product (RNA or protein) may be isolatedand/or detected by methods well known in the art. Following detection,one may compare the results seen in a given cell line or individual witha statistically significant reference group of non-transformed controlcells. Alternatively, one may compare production of RNA or proteinproducts in cell lines transformed with the same gene operably linked tovarious mutants of a promoter sequence. In this way, it is possible toidentify regulatory regions within a novel promoter sequence by theireffect on the expression of an operably linked gene.

[0293] In certain embodiments, it will be desirable to use genes whoseexpression is naturally linked to a given promoter or other regulatoryelement. For example, a prostate specific promoter may be operablylinked to a gene that is normally expressed in prostate tissues.Alternatively, marker genes may be used for assaying promoter activity.Using, for example, a selectable marker gene or a specific antibody, onecould quantitatively determine the resistance conferred upon a tissueculture cell line or animal cell by a construct comprising theselectable marker gene operably linked to the promoter to be assayed.Alternatively, various tissue culture cell line or animal parts could beexposed to a selective agent and the relative resistance provided inthese parts quantified, thereby providing an estimate of the tissuespecific expression of the promoter.

[0294] Screenable markers constitute another efficient means forquantifying the expression of a given gene. Potentially any screenablemarker could be expressed and the marker gene product quantified,thereby providing an estimate of the efficiency with which the promoterdirects expression of the gene. Quantification can readily be carriedout using either visual means, or, for example, a photon countingdevice.

[0295] A preferred screenable marker gene for use with the currentinvention is β-glucuronidase (GUS). Detection of GUS activity can beperformed histochemically using 5-bromo4-chloro-3-indolyl glucuronide(X-gluc) as the substrate for the GUS enzyme, yielding a blueprecipitate inside of cells containing GUS activity. This assay has beendescribed in detail (Jefferson, 1987). The blue coloration can then bevisually scored, and estimates of expression efficiency therebyprovided. GUS activity also can be determined by immunoblot analysis ora fluorometric GUS specific activity assay (Jefferson, 1987). Otherpreferred screenable marker genes that could be used with the inventioninclude LUC, LacZ or various GFPs.

[0296] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

EXAMPLE 1 Description of Plasmids and Lees

[0297] To construct the inventors' standard reporter plasmid, pCMViLUC,the luciferase-encoding LUC+ gene from pGL3-basic (Promega, Inc.) wasinserted as an MluI/XbaI fragment into expression vector pCMV-5(Andersson et al., 1989), and a chimeric intron from pCI (Promega, Inc.)was added as a SacI/EcoRI fragment.

[0298] The complete 3.4 kb LEE.LUC was built by PCR®-amplifying the CMVipromoter, luciferase coding sequence, and hGH terminator from plasmidtemplate pCMViLUC by standard protocols (Perkin-Elmer, Inc.). Twostandard 20-mer primers were used that correspond to the 5′ and 3′ endsof the promoter and terminator regions, nucleotide positions 151 and1590, respectively, of pCMV-5. The LEE components were built byseparately PCR®-amplifying the CMVi promoter, a coding sequence, or hGHterminator from plasmid templates by standard protocols (Perkin-Elmer,Inc.). Primers were synthesized with standard phosphoramidites except,when indicated, five dU residues were incorporated every third position15 bases from the 5′ end to cause UDG sensitivity. The 1.1 kb CMVipromoter component was amplified from pCMVi. It includes the CMVimmediate early gene promoter from pCMV-5, spanning nucleotide positions151 to 915, and the chimeric intron sequences of pCI (Promega, Inc.)from position 1063 to 722. The reverse primer for the CMV.LEE beginswith a 15 base dU stretch: ACUACUACUACUACU (SEQ ID NO: 1), then 18 basescorresponding to the complement of chimeric intron sequences.

[0299] The LUC gene was amplified from plasmid template pLUC, from whichthe CMV promoter and intron had been deleted. Two different LUC productswere made, one with (2.26 kb) and one without (1.66 kb) the hGHtermination region. Both products included nucleotide positions 85through 1742 of pGL3-basic and the 2.26 kb fragment extended into thetermination sequence of pLUC. The forward primer for both productsbegins with the dU stretch: AGUAGUAGUAGUAGU (SEQ ID NO:2) then continueDwith 18 nucleotides corresponding to the LUC gene. The 5′ end of thereverse primer for the 1.66 kb product begins with a 14 base dU region:AUGAUGAUGAUGAU (SEQ ID NO:3), then continues with 18 nucleotidescorresponding to the component LUC gene. The standard hGH 3′ primer wasused. The 0.61 hGH terminator was amplified from pCMV, spenningnucleotides 980 to 1570. The forward primer for the hGH terminator LEEbegins with a 14 base dU stretch: AUCAUCAUCAUCAU. The standard hGH 3′primer was used.

[0300] Crude PCR® products may be isolated from agarose gels or filteredthrough Qiaquick membranes (Qiagen, Inc.). To produce 3′ overhangs, PCR®products were treated with one to two units of UDG (NEB, Inc.) per μg ofDNA for 30 min at 37° C. in reaction buffer (20 mM Tris-HCl pH 8.0, 1 mMEDTA, 1 mM DTT). The enzyme was heat-inactivated, and the samples wereextracted and ethanol precipitated. Pellets were resuspended inannealing buffer (20 mM Tris-HCl pH 8.4, 1.5 mM MgCl₂) at aconcentration of 0.25 μg/ml, melted briefly at 95° C., then transferredto a 72° C. After the addition of 0.5M KCl, samples were slowly cooledto 25° C. to allow annealing.

EXAMPLE 2 Efficient Expression of a Linear Expression Element in Animals

[0301] An amplification product was produced containing acytomegalovirus promoter (CMVi), a luciferase reporter gene (LUC), andthe human growth hormone gene terminator (hGH). This in vitro-produced,non-replicating LEE was precipitated onto gold microprojectiles and shotwith a gene gun (Sanford et al., 1991) into mouse ears. For comparison,a molar equivalent of the LUC gene with the same promoter and terminatorwas delivered on a supercoiled plasmid (pCMViLUC), and the skin tissueswere assayed for luciferase activity 24 to 36 h later (FIG. 1).

[0302] The standard supercoiled LUC plasmid generated an average of7.3×10⁷ lumens per bombarded ear. Background luminescence from tissuebombarded with empty plasmid (pCMVi) was no higher than untreated tissueor sample blanks.

[0303] Reporter gene activity produced from a crude PCR® reactioncontaining the product that encodes luciferase (LEE.LUC) was 19% of thestandard activity. Gel-isolation of the LEE.LUC product before deliveryimproved subsequent gene activity to 67% of that produced fromintroduction of the standard plasmid, and a one-step filter purificationraised LEE.LUC generated activity to near that of the plasmid (93%).Contaminating template contributed insignificantly to the measured geneexpression since introducing a sample that had been treated in exactlythe same manner as the filter-purified sample, but without Taqpolymerase, led to only 0.5% of the luciferase activity. Primers,unincorporated nucleotides, or electrophoresis contaminants in theunpurified LEE.LUC samples may decrease the efficiency of genetransfection or expression. Since these impurities are easily avoided,subsequent studies were conducted with filter-purified PCR® products.

EXAMPLE 3 Production of Linked LEES

[0304] The data reported in Example 2 demonstrate that gene expressionlevels from a PCR® product encoding a complete gene are similar to thatfrom a conventional plasmid. However, while direct amplification ofwhole genes will be useful for a number of applications, it will notalways be possible to amplify contiguous promoter, ORF, and terminatorsequences. For example, most gene screening assays will require fusing acommon promoter to a variety of ORFs, or a reporter ORF to a variety ofpromoters. Screening microbial genes in mammalian cells would requirefusing the prokaryotic ORFs to a eukaryotic promoter and terminator.Therefore, an ideal LEE system will typically involve amplification ofonly an ORF which would then be linked to a set of standard promoter andterminator sequences.

[0305] To resolve the above issues, the inventors developed a strategyto produce complementary single-stranded overhangs on the ends ofseparate PCR® products carrying the ORF, promoter, and terminatorsequences. Single-stranded regions of sufficient length anneal and theattached expression components are directly delivered into tissue.

[0306] A number of methods to create sequence-specific single-strandedoverhangs on any PCR® product were investigated The most efficientprocedure was to prime the chain reactions with oligonucleotides thatbegin with a 12 to 15 base complementary stretch in which deoxyuridines(dU) are incorporated every third position. After amplification, theproducts were treated with uracil DNA glycosylase (UDG) to excise uracilresidues (Nisson et al., 1991). The resulting abasic sites destabilizestrand-annealing within the primer regions. The remaining 3′ strands areintact and provide complementary overhangs by which the separate PCR®products can link. (FIG. 2).

[0307] For example, to build the antigen-expressing LEE.AAT, which isdiscussed in Example 7, the AAT gene-coding sequence (Andersson et al.,1989) was amplified in cis with the hGH terminator sequence describedabove, using pCMViAAT as template. A standard antigen expressionplasmid, pCMViAAT, contains the human AAT gene from pCMVAAT (Tang etal., 1992) cloned as an EcoRI fragment into pCMVi. The 5′ end of theforward primer for the 1.9 kb PCR® product contained dUs:AGUAGUAGUAGUAGU (SEQ ID NO:2) and the 3′ terminator primer was standard.

[0308] Thus, LEEs can be built by amplifying the ORFs from start to stopcodon using DNA as template. The 5′ primers may begin with a uracil richstreach such as, for exaple, AGUAGUAGUAGUAGU (SEQ ID NO:2) then continuewith ORF-specific nucleotides retrieved from, for example, the GenBankdatabase. The 3′ primers begin with another uracil rich streach, forexample, AUGAUGAUGAUGAU (SEQ ID NO:3) then continue with an ORF-specificsequence. These dU ends are compatible for hybridization to the promoterand terminator sequences described above.

[0309] An alternate promoter sequence may be built in order to includethe other sequences, such as for example, those encoding the secretoryleader of tissue plasminogen activator (tPA) protein (Fisher et al.,1985). The PCR® template for this promoter may be, for example, thegenetic immunization vector pCMVitPA, which was constructed by insertingthe leader sequence as an EcoRI/Bgl2 fragment into pCMVi. Int iscontemplated that other changes will be made to make the 3′ promoterprimer more compatable, such as ACUACUACUACUACU (SEQ ID NO:4) followedby 20 nucleotides corresponding to SEQ ID NO:1, the complement of thetPA leader sequence. Annealing of this alternate promoter to the ORFswill encode the antigens fused to the leader peptide.

[0310] Crude PCR® products may be isolated from agarose gels or filteredthrough Qiaquick membranes (Qiagen, Inc.). To produce 3′ overhangs, PCR®products can be treated with one to two units of UDG (NEB, Inc.) per μgof DNA for 30 min at 37° C. in reaction buffer (20 mM Tris-HCl pH 8.0, 1mM EDTA, 1 mM DTT). The enzyme can be heat-inactivated, and the sampleswere extracted and ethanol precipitated. Pellets can be resuspended inannealing buffer (20 mM Tris-HCl pH 8.4, 1.5 mM MgCl₂) at aconcentration of 0.25 μg/ml, melted briefly at 95° C., then transferredto a 72° C. After the addition of 0.5M KCl, samples can be slowly cooledto 25° C. to allow annealing.

[0311] LEEs can be prepared for gene-gun delivery by directly addingPCR® products to the gold precipitation reactions without priorannealing. Annealing efficiency can be determined by gelelectrophoresis, typically yielding 50%-100% full length products. Forcalculating reporter and antigen gene doses, LEE annealing may beassumed to be 100%. Therefore all values will reflect expression from aconservatively estimated number of full length LEEs.

EXAMPLE 4 Noncovalently Linked LEES Express in vivo

[0312] To determine whether the noncovalently linked LEEs producedaccording to Example 3 can express genes in vivo, the CMVi promoter anda luciferase reporter gene (including a terminator) were separatelyamplified using one standard and one dU-containing primer in eachreaction. Annealing of the Uracil Deglycosolase (UDG)-treated PCR®products carrying either a CMVi promoter or a LUC gene to form acomplete LEE (bi-LEE.LUC) with 1 unit or 2 units of UDG. The twoproducts electrophoresed as expected for 1.1 kb and 2.3 kb fragments.These were incubated with UDG and annealed to form the 3.4 kb expressionelement bi-LEE.LUC.

[0313] Reporter genes were introduced into the ears of Swiss-Webstermice with a gene gun (Sanford et al., 1991) (built by Rumsey-Loomis,Ithaca, N.Y.) or with a needle into the tibialis anterior muscle (Wolffet al., 1990). An equal gene-dose of luciferase reporter gene was borneon LEE (3.4 kb) and plasmid (6.7 kb). Ear and muscle tissues wereharvested one to two days later and assayed for activity with standardreagents (Promega, Inc.). Readouts were normalized to 1.0 μg plasmid and0.5 μg LEE. Protein determinations done on lysates in several studiesshowed that any group of muscles, or group of ears, contained similarprotein levels but the total amount of protein extracted from musclesamples was generally twice that extracted from ear samples. However forsimplicity, activities are reported as total lumens per tissue, whichmay underestimate expression per cell from gene-gun delivered samples.The graph in FIG. 3 shows that biolistic introduction of the unligatedsample into mouse skin produced 85% of the luciferase activity producedfrom delivery of an equivalent gene-dose of the LUC plasmid, assumingcomplete annealing of the LEE.

[0314] Background expression due to PCR®-template contamination wasprevented by using plasmid templates that were LUC-less or promoter-lessto amplify the promoter or LUC gene, respectively. As expected, controlsamples without polymerase showed no reporter activity. Resultsindicated that attachment of a promoter and ORF by simpleoverhang-hybridization enables plasmid-like levels of gene delivery andexpression of an LEE in the animal.

[0315] To test the importance of fragment annealing for generating thisgene expression, the dU primed PCR® products were introduced withoutbeing pre-treated with UDG. Surprisingly, these LEEs produced 29% of thestandard-plasmid reporter-gene activity. The relatively high level ofgene expression suggested that exposure to endogenous murine UDG (Nissonet al., 1991) enables in vivo annealing and sticky-end ligation.

[0316] The promoter and reporter gene were also amplified with standardprimers that did not contain dUs. Introduction of these blunt-ended PCR®fragments produced low but detectable levels of luciferase activity,presumably due to in vivo blunt-end ligation. Either product deliveredinto separate animals produced no reporter activity. (FIG. 3).

EXAMPLE 5 Production of LEES with Terminator Sequences

[0317] Many applications of the LEE technique would require attachingboth a new promoter and a terminator to a particular coding sequence,for example, in cases where a native terminator is not included with theORF. Therefore, the practicality of such a linkage was assessed. Athree-component LEE expressing luciferase (tri-LEE.LUC) was preparedfrom PCR® products that separately carried the CMVi promoter, LUC codingsequence, and hGH terminator. The dU primed amplification products wereUDG treated, annealed by strand-hybridization without ligase, andimmediately shot into mice. The data in FIG. 4 show that the level ofluciferase activity generated by this triple arrangement was 77% of thatfrom a single PCR® product carrying all three components, whereas,delivery of an LEE composed of a promoter and ORF but not a terminatorproduces negligible gene activity. This is impressive since the yield offully annealed product would presumably decrease as the number ofrequired interactions increase.

EXAMPLE 6 Production of CEES

[0318] A circular expression element (CEE) was engineered to simplifythese gene vehicles. This dU-primed PCR® product carries adivergently-oriented promoter and terminator at its ends so that thiselement can be linked to an ORF to add both expression sequences to anORF. The resulting circular element is not a plasmid since it isnonreplicative and contains only the sequences necessary for the desiredgene expression. Other genes in CEEs, like LEEs, are effectivelydelivered in vivo to yield reporter gene activity. In particularembodiments there is not excess additional superfluous plasmid backbonesequences comprising a CEE. In particular aspects the CEE is produced byPCR® amplification.

EXAMPLE 7 LEES Introduced into Animals are Expressed and Result inAntibody Expression

[0319] To test the ability of these linear DNAs to raise an immuneresponse, a LEE was designed to express human α-1 antitrypsin (AAT) bynoncovalently linking the PCR®-amplified CMVi promoter to the AAT ORF(with terminator). The antibody levels presented in FIG. 5 show thatintroduction into mouse skin of either the bimolecular LEE (bi-LEE.AAT)or a molar-equivalent of a standard supercoiled plasmid (pCMViAAT)produced AAT-specific responses. Antibody titers in the two AAT-genevaccinated groups were similar and all five mice per group responded. Acontrol group vaccinated with empty plasmid did not produce a response.

[0320] Groups of five BALB/c mice were vaccinated with the antigen geneAAT on a plasmid or LEE, or with a control plasmid. One μg pCMViAAT, 0.5μg bi-LEE.AAT or 1 μg pCMVi were biolistically delivered into the ears.Animals were boosted at week two and bled four months later withoutfurther boosts. Sera from groups were individually collected and testedin duplicate for AAT-specific antibodies by ELISA using standardprotocols (Harlow and Lane, 1988). Primary sera were diluted 1:500, 1:1000, and 1:2000 in TBS-T (Harlow and Lane, 1988) and 2.5% BSA. Data inFIG. 5 are reported from sera diluted 1:1000. Concentrations ofAAT-specific IgGs in sera were determined by serially diluting amonoclonal anti-AAT antibody (CalBiochem, Inc.).

EXAMPLE 8 Genetic Immunizations Employing LEES and CEES

[0321] LEE and CEE methods are well-tailored for genetic immunizations(Tang et al., 1992). Each ORF of a pathogen could be attached toeukaryotic expression sequences, introduced into a host, and tested as aprotective antigen. In addition, any ORF could be introduced into miceor rabbits to produce antibodies for analytical purposes (Barry et al,1994).

[0322] To directly demonstrate the potentials of this protocol forproducing useful antibody reagents or for the development of animportant vaccine, LEEs may be built to express pathogen ORFs. ORFs canbe amplified from genomic DNA with dU primers, UDG-treated and linked toa mammalian promoter and terminator as described. Each LEE can be usedto vaccinate an organism, such as sets of BALB/c mice; a plasmidencoding murine GMCSF can be included as a genetic adjuvant.

[0323] The LEEs expressing ORFs can be used to biolistically immunize anorganism, such as for example, BALB/c mice. 2 μg of each LEE encoding anORF can be shot into the ears of two mice. Each set of animals can beboosted at weeks three and five then bled twelve days later.

[0324] Sera from immunized and non-immunized mice can be subsequentlyanalyzed. A standard immunoblot can be used to analyze the sera againsttotal cell lysates of the ORF's originating organism (20 μg)gel-fractionated in 12.5% SDS-PAGE (Harlow and Lane, 1988) and the serafrom mice that were vaccinated biolistically with 2 μg of ORF can beanalyzed by immunoblot. Sera can be diluted, for example, 1:300 in TBS-Tand 5% dry milk. HRP-conjugated rabbit antimouse IgG+IgM (Pierce, Inc.),or other suitable secondary antibody can be diluted, for example, in1:2500 in TBS-T and 5% milk and used as a secondary antibody forchemiluminescent (Renaissance, NEN, Inc.) detection. Unimmunized but agematched mice are expected to show no specific reactivities.

[0325] Vaccination with the LEEs are expected to generate antibodies inthe animals that recognized specific polypeptides. It is expected thatunpropagated, unligated PCR® products can be used to produce specifichumoral immune responses against relevant pathogens.

[0326] It is contemplated that LEEs can be used as gene vaccines toraise antibodies against encoded eukaryotic or prokaryotic antigens.Sufficient gene expression is expected to be achieved in order tostimulate humoral responses.

EXAMPLE 9 Expression of LEES and CEES in Tissue Culture

[0327] The LEE and CEE technology may also be used for cell culturetransfections. The performance of the reporter gene LEEs was tested inPEA10 mouse fibroblasts. For cell culture transfections, the sameluciferase reporter gene was delivered into murine, BALB/c-derivedfibroblast cells (PEA10) with a chamber gene-gun (Roomsey-Loomis,Ithaca, N.Y.). Plates of 10⁶ cells were shot in triplicate with equalgene-doses of LEE.LUC or LUC plasmid. Cells were harvested into lysisbuffer 18 h later. Total protein concentrations were determined (Pierce,Inc.) for each sample. Luciferase activity was measured and calculatedas lumens per mg lysate.

[0328] The standard pCMViLUC plasmid produced 4.8×10⁶ lumens permilligram of cell lysate while the empty pCMVi plasmid led to noluminescence above sample blanks. (FIG. 6) Luciferase activity generatedby the full length LEE.LUC PCR® product was 35% of the standard plasmid.The linked biLEE.LUC and tri.LEE.LUC products expressed 31% and 21%,respectively, of the plasmid-encoded reporter gene activity. Inparallel, mice ears were transfected with the same preparations ofeither pCMViLUC or full length LEE.LUC and assayed 18 h later.Luciferase activity produced by the LEE was 34% of the standard plasmid.These somewhat lower LEE expression levels both in vitro and in vivo maybe a result of a dirtier LEE preparation of the shorter time pointassayed. Nonetheless, these results demonstrate a direct correlationbetween the gene expression levels produced by LEEs transfected intocell cultures with those produced by their transfection into animals.

EXAMPLE 10 Utilization of LEES and CEES to Functionally Screen ViralGenes

[0329] LEEs or CEEs can be used to functionally screen a genome for agene or gene fragment that encodes a product with any activity orindirectly causes an activity that can be assayed. For example, all theopen reading frames (ORFs) of a sequenced genome can be PCR-amplifiedwith dU-containing primers directed to the coding sequence endpoints(translation start and stop codons). These ORFs can be linked topromoter- and terminator-containing PCR products as described earlier tocreate a LEE library. The library can be split into sub-libraries forscreening. The number of LEEs per sublibrary and the total number ofsublibraries can be varied to accommodate the assay conditions. DNAcorresponding to each LEE sublibrary can be delivered biolistically intotissue cultures or into the skin of animals. Transient gene expression,physiological activities and effects, or immune activities and responsescould be assayed. Positively scoring sublibraries could be further splitin order to test less complex sublibraries, reiteratively, until asingle LEE or set of LEEs is identified.

[0330] For example, LEEs can be used in the functional screening ofviral genes for accumulation of dendritic cells in skin. Individualviral genes can be amplified by polymerase chain reaction (PCR®) usingdU-containing primers. Groups composed of 8-10 PCR® products can bemixed with, for example, CMV promoter and hGH terminator sequencesamplified with compatible dU-flanks. After treatment with UDG andannealing, the DNA can be precipitated onto gold particles andintroduced into skin of mice with the gene gun. After 4 days, the skincan be harvested, and thin sections prepared and stained for thepresence of dendritic cells with anti-1^(a) antibody. An experimentalgroup and a luciferase would not be exprected to show a net accumulationof dendritic cells, alongside experimental group X that shows a netaccumulation of dendritic cells in the area surrounding the goldparticles. Group X can be further split into 10 groups of a single LEEeach, these can be introduced into the skin of mice. This enabled asingle LEE, expressing one viral ORF to be identified that causesdenditic cell recruitment.

EXAMPLE 11 Methods of Employing LEES and CEES to Produce Vaccines

[0331] Each pathogen ORF can be generated, annealed to a mammalianpromoter and terminator then directly introduced into a test animal as avaccine. The linear expression elements (LEEs) can be screenedindividually or in pools. With this method it is envisioned that all thegenes of a pathogen can be introduced as genetic vaccines into animalsin a matter of days. The animals can be subsequently challenged withpathogen to determine which genes protect against disease. Isolatingindividual LEEs from protective pools can be conducted as previouslydescribed for plasmid libraries (Barry et al, 1995).

[0332] In a preferred embodiment, the ORFs of a pathogen are amplifiedby PCR® for expression by LEEs. Such ORFs may be from known genesequences deposited with a genetic database, such as for example, theNational Center for Biotechnology Information's Genbank and GenPeptdatabases (http://www.ncbi.nlm.nih.gov). The coding regions for theseknown genes may be amplified and/or expressed using the techniquesdisclosed herein. Additionally, genes may be amplified using LEEs from acommercially available genetic library specific to a particularpathogen. Or a genetic library specific to one or more pathogens may beprepared, for genomic or cDNA sequence, as would be known to one ofordinary skill in the art (Sambrook et al. 1989). The genes could alsobe chemically synthesized as linear elements for direct introductioninto animals.

[0333] A typical procedure would be to synthesize oligonucleotides forthe amplification of each gene in a sequenced genome. These oligos wouldbe designed such that they would be used to create LEEs or CEEs asdescribed. ORFs of each gene would be PCR® amplified and the CMVpromoter and human growth hormone terminator linked as described. TheLEEs could be pooled in groups of ˜50, for example, and introduced intothe skin by a gene gun or injected with a needle into the test animal.These animals would receive one boost ˜3 weeks later of the same DNA andthen challenged with the pathogen some time after this. If a group ofanimals showed resistance to infection, the ˜50 genes in that pool wouldbe tested individually or in smaller groups to isolate the onesresponsible for protection. If the thousands of genes of the pathogenwere cloned by conventional practice, the creation of the pools forscreening would have taken months as opposed to a week using LEEs orCEEs.

[0334] If the genome sequence was not available, the genome could besubjected to random amplification with dU adapter oligos. These randomlyamplified fragments would be linked to the CMV promoter and terminatoras before and then pooled for testing as vaccines. Only ˜⅙th of thefragments would encode real ORFs.

[0335] It is envisioned that LEE technology should permit the rapidscreening of all the genomes of pathogens for vaccine candidates.

[0336] For example, LEE technology was used to generate random LEElibraries from pathogen genomes designed for the subsequent screening ofgenes and gene fragments for vaccine candidates. No sequence database isnecessary. Instead of using dU-containing primers directed to knownORFs, dU-containing random primers were used. Random LEE libraries havebeen made from the genomes of Herpes Simplex Virus (a large virus) andMycoplasma pulmonis (bacteria), or a parasite. The genomicrepresentation of the LEE libraries appears to be broad as measured bythe high complexity of the PCR products. The genetic representation wassampled by the successful amplification of a number of known genefragments from the libraries. Although only ⅙^(th) of the LEEs wouldproperly express a coding region, due to frame and orientationconsiderations, the ease of production means that very large complexitylibraries could be generated. Genomically complete libraries could begenerated even from large pathogenic genomes such as parasites forvaccine screening.

EXAMPLE 12 Methods of LEES and CEES to Develop Immunological Reagents

[0337] The LEE/CEE approach could be used to rapidly produceimmunological reagents such as antibodies. In a representative example,the ORF of one or more genes or portions of genes would be PCR®amplified or chemically synthesized with adapter sequences such that itwould attach to a promoter and terminator. These LEEs would then bedirectly introducted in to an animal to create an immune response to theencoded gene product, for example antibodies. These antibodies could beused directly for research purposes to study that particular gene, forexample, which genes are expressed in a particular stage of a pathogento create vaccine candidates. This procedure could also be used todiscover antibodies or other immunological reagents that are useful indiagnostic procedures or have therapeutic value. Since it is very easyto create LEEs and immunize animals with them to create an immuneresponse, it should be possible to rapidly screen many genomes forproduction of immunologically useful reagents.

[0338] LEEs and CEEs can be used in developing immunological reagentsfor vaccine identification. LEEs and CEEs can be used to produceantibodies to an ORF. Antibodies to all a pathogen's ORFs can beproduced, then used to probe pathogen-infected tissue byimmunohistochemistry to elucidate which proteins are present in aparticular tissue at any pathogen stage-of-interest. Identified geneproducts are excellent vaccine candidates or drug targets.

[0339] In a non-limiting example, useful immunological reagents weregenerated by LEE technology for the identification of vaccine candidatesagainst the parasite Plasmodium falciparum, the etiological agent ofMalaria. A family of diverse genes, called Rifins, with predictedcell-surface localizations was identified following the sequencing ofthe first chromosome from Plasmodium. Gene coding regions from 16 Rifinswere PCR-amplified, linked to mammalian promoter and terminator, thenused as inocula to vaccinate groups of CD1 mice for anti-Rifin antibodyproduction. Sera from blood samples were used to probe liver-tissuesamples from Plasmodia-infected mice using immunofluorescence assays.From the 16 randomly chosen probes, three positives were found. Thisdemonstrates that the LEE inoculum used to generate the positive seraencodes a protein that exists in the liver-stage disease. Since thepresence of these proteins in the liver makes them likely vaccinecandidates, the LEE screen may have dramatically streamlined the searchfor candidates.

[0340] In another non-limiting example, a LEE ORF library has been madethat encodes all the ORFs of Herpes Simplex Virus (HSV). The genomic andgenetic representations have been tested as described. Each LEE has beendelivered into a pair of CDI mice for antibody production against theLEE-encoded gene product. Sera will be harvested from the animals toobtain a complete repertoire of HSV antibodies. This type of reagent setwill be useful for both clinical and research applications such asproteomics. Similar LEE or CEE libraries could be generated from othergenomes of interest.

EXAMPLE 13 Methods of Using LEE and CEE Technology to Screen forPromoter Function

[0341] By reversing the variable and constant portions of an LEE/CEE itshould be possible to screen for a particular promoter or otherregulatory functions. For example, the promoters of many sequenced genescould be PCR® amplified such that they are LEE elements and attached toa constant reporter (e.g., GFP, Luc, B-gal) ORF. These LEEs could thenbe shot into, for example, brain sections, and these sections thenassayed for which LEE or group of LEEs produced reporter gene product ina particular cell type, e.g., neurons. In this way, the cell specificregulation pattern of any promoter could be determined readily, or largenumbers of promoters assayed for expression in particular cell types.

EXAMPLE 14 Use of LEE and CEE Technology to Test for Biological Functionof ORFS

[0342] In another application, the genes of a virus can be transformedinto LEEs, and these put, for example, in 26 groups of 8-10 LEEs. Thegroups can be shot into the adominal skin of mice, and the skin observedone day later for the number of dendritic cells in the bombarded area.In this way, a group of genes that affected dendritic cell migration canbe identified. By using the LEEs, this screening could be accomplishedin days, where if conventional cloning had been relied on to isolate thelarge number of genes in a viral genome, it is expected to take months.Other immune related and unrelated biological activities could bescreened such cytokine production or enzyme production.

[0343] It is envisioned that it should be possible to screen for anassayable biological function of gene products by directly introducingLEEs/CEEs into organisms, tissue sections or cells. By directobservation of effects on particular cell types versus other cell types,it should be possible to efficiently isolate proteins that have cellspecific effects.

EXAMPLE 15 Use of LEES/CEES to Screen Gene Products in Cell-Free Systems

[0344] It would be very easy to attach an ORF or many ORFs from a genomeor other sequence database to a promoter which facilitates in vitrotranscription and translation in a cell-free system. Such a construct,for example, with the T7 polymerase promoter, could be used to rapidlyproduce proteins corresponding to many different genes. This proteincould be radioactively or otherwise tagged. These tagged proteins couldthen be purified or directly screened for those that had a particularfunction, for example, were able to bind to another particular proteinor drug or even that had a particular enzyme activity. This applicationof the LEE/CEE technology could greatly enhance the proteomic effort,that is, to screen all the proteins of genomes for particular functionsor uses.

[0345] A particular example of using LEE expressed gene products thatare in vitro produced is for the isolation of antibodies orantibody-like molecules. Antibodies are key tools for proteomicanalyses. Complete repertoires of antibodies that bind all the proteinsof an organism are highly desirable. In a high throughput fashion, LEE'scould be synthesized in vitro, products purified then used to fish out,for example, reactive single-chain antibodies expressed within asingle-chain-antibody phagemid library. The diversity within theartificial antibody library is much greater than possible in animals,and the time required for the experiment is considerably shorter,requiring no animals.

EXAMPLE 16 Expression of LEES/CEES in Plants

[0346] The LEE and CEE technology may also be used for expression ofgenes in plant protoplasts and whole plants. For example, the strong 35Spromoter sequence from Cauliflower Mosaic Virus (CaMV) could bePCR-amplified, as described ealier, with a standard 5′-end primer and adU-containing primer at its 3′end. The plant reporter genebeta-glucuronidase (GUS) could be amplified with two dU-containingprimers. The efficient termination sequence from the nopaline synthetase(NOS) could be amplified with a dU-containing 5′end primer and astandard 3′end primer. UDG treatment would yield single-stranded endsthat could anneal that to form a fully functional GUS gene expressionunit. The LEE can be precipitated on gold microparticles and deliveredwith a gene gun into plant cells, tissue, or whole samples. A standardGUS reporter assay will determine gene expression (Jefferson et. al.(1987) EMBO J., pp3901-3907).

EXAMPLE 17 Use of LEES/CEES to Test Plant ORFS for Biological Functionsof Interest

[0347] The same amplification procedure described in example 16 can beused to generate a LEE that expresses any plant gene of unknown orputative function. Plant-transformation will enable very fast analysisof in vivo gene function. Plant genomes are being sequenced, such asArabidopsis, and this is identifying new ORFs without know functions.Many times these ORFs have sequence similarity to genes with knownfunctions. The activities of the possible homologues can be quicklytested by constructing LEEs that encode the new ORFs then assaying themin vitro or in vivo for the supposed activity.

[0348] LEEs with plant ORFs could be constructed and introduced intoplant tissue directly to screen for anything that could be screened foras a local or individual cell trait, or introduce the LEE into cellcultures and screen for cells that display individual trait like diseaseor herbicide resistance; or shoot all ORFs into plant cells to generateindividual transgenic plants to screen for any trait including wholeplant features, such as for example, yield or drought resistance.

[0349] In particular, the LEE technology could be used to screen plantopen-reading frames for useful biological functions. For example, thecommonly used 35S promoter or other plant promoters could be PCRamplified with ends that could anneal with the overhangs of the PCRproducts of each ORF. In the same manner the 3′ ends of the ORF may bedesigned to anneal with a common plant terminator sequence, for examplethe nopaline synthetase (NOS) termination sequence. These ORFs could bescreened for useful functions by introducing them directly into plantsor into plant cell cultures. For example the ORFs could be introducedinto cell cultures and which were then challenged by a pathogen toscreen for ORFs that confer resistance to infection, the ORFs could bescreened for ones that conferred protection against herbicides, or theLEE ORFs could be introduced into cells to produce callus andsubsequently transgenic plants to then screen for effects on yield,disease resistance, cold or heat resistance, stature, daylenghtresponse, or nutritional trait. In this case the LEE would beco-introduced with a selectable marker or with a selectable trait, forexample kanamycin resistance, incorporated into the LEE.

EXAMPLE 18 Use of LEES/CEES to Test Plant Promoters

[0350] A plant reporter gene as described above can be linked todifferent promoters, delivered into plants, then tested for tissue orstage-specific activity, or for enhanced promoter activity. In coulddesirable to express a gene normally expressed in one tissue in adifferent tissue, or to increase the expression of an endogenous gene.For example pathogens can often infect one but not another tissue. Ifthe gene enabling the resisitance of one tissue can be identified thenengineered with a promoter expressed in the sensitive tissue, then plantprotection may be achieved. Increasing the expression of a gene relatedto fragrance or color may improve the value of a flowering plant.

[0351] All predicted or suspected promoters from a plant could be linkedto a reporter gene (for example GUS) and terminator and introduced intothe whole plant or tissues, cell cultures or cells to make transgenicplants. The activity of the promoters could then be assayed for usefultraits. For example, all the promoters could be linked to GUS by LEE andintroduced into roots to find promoters that express preferentially andstrongly in roots. Or the promoter -GUS LEEs could be introduced intoplant cell cultures and the cells exposed to an enviromental agent (forexample, heat, cold, salt, light, herbicides) to determine whichpromoters respond to which stimuli. Alternatively, transgenic plantscould be generated using LEEs as described above where the promoterswere linked to a particular ORF and the transgenic plants screened for adesired trait. For example, all promoters could be linked to aheat-shock protein and the resulting transgenic plants screened for heattolerance. The ORFs or promoters sceeened in plants could be from oneplant species screened in another. For example all the ORF of a diseaseresistant plant could be screened in a susceptible plant. The ORFs orpromoters could be from non-plants and transfected into plants.

EXAMPLE 19 Use of LEES/CEES to Generate Plant Libraries for theFunctional Screening of Genes or ORFS of Interest

[0352] LEE libraries can be generated that encode plant genes, genefragments, or ORFs. These can be transformed as groups into plants toscreen for an activity of interest, including protection from plantpathogens. Positively scoring groups can be reduced as described earlieruntil single ORFs with the activity of interest are isolated. If theplant genomic sequence is not available, random plant libraries couldalso be generated and tested. For example, one plant species may beresistant to infection from a pathogen. A distinct species, perhapsrelated but to necessarily related, may be used in crops but ispathogen-sensitive. It would be possible to prepare a LEE librarycorresponding to all the ORFs of the resistant plant then deliver themin groups into the sensitive plant species. Pathogenic challenge of thenormally sensitive plants would identify plants that express the foreignresistance gene(s). Sibbing and further screening could isolate singleORFs.

EXAMPLE 20 Expression of LEES/CEES in Brain or Other Tissue Sections

[0353] LEEs could be delivered into live tissue sections such as ratbrain slices. Many different promoters could be linked to a reportergene and terminator. Comparison of reporter activity in the tissue ofinterest, such as brain, to a control tissue would quickly identifytissue-specific promoters. Alternatively, promoters could be leftattached to their natural genes, delivered as LEEs, and tissue-specificexpression could be asssesed by visualizing the LEE-encodedgene-products with specific antibodies. As another application, theintracellular localization of the encoded gene product could bedetermined by linking a gene of interest to a standard promoter, such asCMV, then visualizing the LEE-encoded product by immunohistochemistry.

[0354] All of the compositions and methods disclosed and claimed hereincan be made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and methods and in the steps or in the sequence ofsteps of the methods described herein without departing from theconcept, spirit and scope of the invention. More specifically, it willbe apparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

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1 5 1 15 RNA Artificial Sequence Description of Artificial SequenceSynthetic Primer 1 acuacuacua cuacu 15 2 15 RNA Artificial SequenceDescription of Artificial Sequence Synthetic Primer 2 aguaguagua guagu15 3 14 RNA Artificial Sequence Description of Artificial SequenceSynthetic Primer 3 augaugauga ugau 14 4 15 RNA Artificial SequenceDescription of Artificial Sequence Synthetic Primer 4 acuacuacua cuacu15 5 14 RNA Artificial Sequence Description of Artificial SequenceSynthetic Primer 5 aucaucauca ucau 14

What is claimed is:
 1. A method of assaying for the production orregulation of expression of at least one polypeptide from a linear orcircular nucleic acid segment comprising a promoter or putative promoterand an open reading frame or putative open reading frame, the methodcomprising: a) obtaining at least one linear or circular nucleic acidsegment comprising a promoter or putative promoter and an open readingframe encoding a peptide or putative open reading frame; b) placing thenucleic acid segment under conditions conducive to expression of thepolypeptide from the open reading frame; and c) assaying for theproduction or regulation of expression of a polypeptide from the openreading frame or putative open reading frame.
 2. The method of claim 1,wherein the nucleic acid segment further comprises a terminator.
 3. Themethod of claim 1, wherein the nucleic acid segment is obtained bysynthesis.
 4. The method of claim 3, wherein the synthesis comprisesnon-covalent linkage of the promoter to the open reading frame.
 5. Themethod of claim 4, wherein the non-covalent linkage is performed by: a)obtaining a PCR® product comprising the nucleic acid segment, which PCR®product is obtained by amplification from at least one primer that hascomplementary stretches comprising deoxyuridines with uracil-DNAglycosylase to create overhangs to which the promoter can link; b)providing a promoter; and c) non-covalently linking the promoter to thenucleic acid segment to create the linear or circular expressionelement.
 6. The method of claim 5, further comprising non-covalentlinkage of a terminator to the open reading frame.
 7. The method ofclaim 5, wherein the primer that has complementary stretches comprisingdeoxyuridines comprises the promoter and the terminator in divergentorientation, such that the step of non-covalently linking the promoterand the terminator to the open reading frame results in a circularexpression element.
 8. The method of claim 1, wherein the nucleic acidsegment is obtained by PCR®.
 9. The method of claim 1, wherein thenucleic acid segment is a linear nucleic acid that is cut out of aplasmid.
 10. The method of claim 1, wherein the nucleic acid segment isplaced into a cell.
 11. The method of claim 10, wherein the nucleic acidsegment is not integrated into the cell's genome.
 12. The method ofclaim 10, wherein the cell is in cell culture.
 13. The method of claim10, wherein the cell is comprised in an organism.
 14. The method ofclaim 13, wherein the organism is a mammal.
 15. The method of claim 13,wherein the organism is a plant.
 16. The method of claim 10, wherein thelinear nucleic acid is injected into the cell.
 17. The method of claim16, wherein said injection comprises microprojectile bombardment. 18.The method of claim 10, wherein the cell is a plant cell.
 19. The methodof claim 10, wherein the cell is an animal cell.
 20. The method of claim1, wherein the nucleic acid segment is placed in a cell-free expressionreaction.
 21. The method of claim 1, further comprising assaying forexpression of the polypeptide.
 22. The method of claim 21, comprisingassaying the expression of a reporter gene product encoded in an openreading frame.
 23. The method of claim 1, comprising assaying forfunction of the promoter or putative promoter.
 24. The method of claim23, wherein assaying for function of the promoter or putative promotercomprises determining whether a reporter gene product encoded in an openreading frame is expressed.
 25. The method of claim 24, comprisingcomparing the function of two or more putative promoters.
 26. The methodof claim 1, wherein the nucleic acid segment is a linear nucleic acidsegment.
 27. The method of claim 1, wherein the nucleic acid segment isa circular nucleic acid segment.
 28. A method of analyzing a nucleicacid sequence comprising: a) obtaining a nucleic acid segment; b)linking the nucleic acid segment to a promoter and a terminator tocreate a linear or circular expression element; c) providing the linearor circular expression element to a cell-free expression system or to acell under conditions conducive to expression of any product encoded forby the nucleic acid segment; and d) analyzing any expression of anyproduct encoded by the nucleic acid sequence.
 29. The method of claim28, wherein the nucleic acid segment is non-covalently linked to thepromoter.
 30. The method of claim 29, wherein the non-covalent linkageis performed by: a) obtaining a PCR® product comprising the nucleic acidsegment, which PCR® product is obtained by amplification from at leastone primer that has complementary stretches comprising deoxyuridineswith uracil-DNA glycosylase to create overhangs to which the promotercan link; b) providing a promoter; and c) non-covalently linking thepromoter to the nucleic acid segment to create the linear or circularexpression element.
 31. The method of claim 30, further comprisingnon-covalent linkage of a terminator to the open reading frame.
 32. Themethod of claim 28, wherein the nucleic acid sequence comprises an openreading frame.
 33. The method of claim 28, wherein more than onedistinct nucleic acid segment is analyzed in a single procedure.
 34. Themethod of claim 28, wherein the linear or circular expression element isplaced into a cell.
 35. The method of claim 34, wherein the linear orcircular expression element is not integrated into the cell's genome.36. The method of claim 34, wherein the cell is comprised in anorganism.
 37. The method of claim 34, wherein the linear or circularexpression element is injected into the cell.
 38. The method of claim37, wherein said injection comprises microprojectile bombardment. 39.The method of claim 28, wherein the linear or circular expressionelement is placed in a cell-free expression reaction.
 40. A method ofanalyzing a nucleic acid segment for activity as a promoter comprising:a) obtaining a nucleic acid segment encoding a putative promoter; b)linking the nucleic acid segment encoding the putative promoter to anucleic acid encoding a polypeptide to create a linear or circularexpression element; c) providing the linear or circular expressionelement to a cell-free expression system or to a cell under conditionsconducive to expression of the polypeptide; and d) analyzing anyexpression of the polypeptide.
 41. The method of claim 40, wherein thenucleic acid segment encoding the putative promoter is non-covalentlylinked to the nucleic acid encoding a polypeptide.
 42. The method ofclaim 41, wherein the non-covalent linkage is performed by: a) obtaininga PCR® product comprising the nucleic acid segment encoding the putativepromoter, which PCR® product is obtained by amplification from at leastone primer that has complementary stretches comprising deoxyuridineswith uracil-DNA glycosylase to create overhangs to which the nucleicacid encoding a polypeptide can link; b) providing the nucleic acidencoding a polypeptide; and c) non-covalently linking the nucleic acidsegment encoding the putative promoter to the nucleic acid encoding apolypeptide to create the linear or circular expression element.
 43. Themethod of claim 40, wherein the nucleic acid encoding the polypeptide islinked to a nucleic acid sequence encoding a terminator.
 44. The methodof claim 40, wherein the nucleic acid encoding the polypeptide encodes areporter gene product.
 45. The method of claim 44, comprising assayingfor expression of the reporter gene product.
 46. The method of claim 40,wherein more than one distinct nucleic acid segment encoding a putativepromoter analyzed in a single procedure.
 47. The method of claim 46,comprising analyzing more than one nucleic acids encoding a putativepromoter.
 48. The method of claim 46, wherein the nucleic acid encodinga putative promoter is a native nucleic acid.
 49. The method of claim46, wherein the nucleic acid encoding a putative promoter was preparedby mutation of a native promoter sequence.
 50. The method of claim 40,wherein the linear or circular expression element is placed into a cell.51. The method of claim 40, wherein the linear or circular expressionelement is not integrated into the cell's genome.
 52. The method ofclaim 50, wherein the linear or circular expression element is injectedinto the cell.
 53. The method of claim 50, wherein the linear orcircular expression element is placed in a cell-free expressionreaction.
 54. A method of screening for a biological responsecomprising: a) obtaining a linear or circular expression element by aprocess comprising: obtaining a DNA segment comprising an open readingframe; linking the open reading frame to a promoter and a terminator tocreate a linear or circular expression element; and b) providing theexpression element to an organism under conditions conducive toexpression of any product encoded for by the open reading frame.
 55. Themethod of claim 54, wherein the DNA segment is obtained from a processinvolving PCR®.
 56. The method of claim 54, wherein the open readingframe is non-covalently linked to the promoter and the terminator. 57.The method of claim 54, wherein the non-covalent linkage is performedby: a) obtaining a PCR® product comprising the open reading frame, whichPCR® product is obtained by amplification from at least one primer thathas complementary stretches comprising deoxyuridines with uracil-DNAglycosylase to create overhangs to which the promoter and terminator canlink; b) providing a promoter and a terminator; and c) non-covalentlylinking the promoter and the terminator to the open reading frame tocreate the linear or circular expression element.
 58. The method ofclaim 54, wherein the linear or circular expression element is injectedinto the organism.
 59. The method of claim 54, wherein more than onetype of linear or circular expression element is introduced to theorganism.
 60. The method of claim 54, further defined as a method ofproducing antibodies for analytical purposes.
 61. The method of claim54, further defined as a method of vaccinating the organism.
 62. Themethod of claim 54, wherein the organism is a mammal or plant.
 63. Amethod of vaccinating an organism comprising: a) obtaining a linear orcircular expression element by a process comprising obtaining a DNAsegment comprising an open reading frame and linking the open readingframe to a promoter and a terminator to create a linear expressionelement; and b) providing the linear expression element to an organismunder conditions conducive to expression of any product encoded for bythe open reading frame, such that immune response is produced in theanimal.
 64. The method of claim 63, wherein the open reading frame isnon-covalently linked to the promoter and the terminator.
 65. The methodof claim 63, wherein the linear or circular expression element isinjected into the organism.
 66. The method of claim 63, wherein morethan one type of linear or circular expression element is introduced tothe organism.
 67. The method of claim 66, wherein a plurality of typesof linear or circular expression elements is introduced to the organism.68. The method of claim 67, wherein the plurality of types of linear orcircular expression elements comprises elements encoding distinctpolypeptides of a pathogen.
 69. The method of claim 68, wherein thepathogen is a virus, bacterium, fungus, alga, protozoan, arthropod,nematode, platyhelminthe, or plant.
 70. The method of claim 68, whereinthe pathogen is a virus.
 71. The method of claim 70, wherein individuallinear or circular expression elements encoding all potential allergensof a virus is comprised in the plurality of types of linear or circularexpression elements.
 72. The method of claim 63, wherein the organism isa human.
 73. The method of claim 63, wherein the organism is a plant.74. The method of claim 63, wherein the open reading frame encodes apolypeptide from a cancer.
 75. A method of selecting open reading frameseffective for generating an immune response specific to a pathogen in anorganism, comprising: a) preparing a plurality of linear or circularexpression elements produced by a method comprising: obtaining pluralityof DNA segments comprising open reading frames from a pathogen; andlinking open reading frames to promoters and terminators to create aplurality of linear or circular expression elements; b) introducing theplurality of linear or circular expression elements into an organism;and c) selecting from the plurality of linear or circular expressionelements open reading frames that are effective to generate said immuneresponse.
 76. The method of claim 75, wherein the pathogen is a virus,bacterium, fungus, alga, protozoan, arthropod, nematode, platyhelminthe,or plant.
 77. The method of claim 75, further comprising testing saidorganism against challenge with the pathogen from which plurality oflinear or circular expression elements was prepared wherein the organismis protected against challenge with the pathogen.
 78. The method ofclaim 77, wherein one or more antigens conferring a protective responseis identified by screening of the organism.
 79. The method of claim 75,wherein the plurality of linear or circular expression elements isinjected into the organism.
 80. A method of producing a linear orcircular expression element comprising: a) obtaining a DNA segmentcomprising an open reading frame; and b) linking the open reading frameto a promoter and a terminator to create a linear or circular expressionelement.
 81. The method of claim 80, wherein the DNA segment is obtainedfrom a process involving PCR®.
 82. The method of claim 81, wherein thePCR® reaction is primed with oligonucleotides having a complementarystretch incorporating deoxyuridines.
 83. The method of claim 82, whereinthe deoxyuridines are incorporated every third position of thecomplementary stretch.
 84. The method of claim 80, wherein the openreading frame is non-covalently linked to the promoter and theterminator.
 85. The method of claim 84, wherein the non-covalent linkageis performed by: a) obtaining a PCR® product comprising the open readingframe, which PCR® product is obtained by amplification from at least oneprimer that has complementary stretches comprising deoxyuridines withuracil-DNA glycosylase to create overhangs to which the promoter andterminator can link; b) providing a promoter and a terminator; c)non-covalently linking the promoter and the terminator to the openreading frame to create the linear or circular expression element. 86.The method of claim 85, wherein the deoxyuridines are incorporated atevery third position of the complementary stretches.
 87. The method ofclaim 85, wherein the primer that has complementary stretches comprisingdeoxyuridines comprises the promoter and the terminator in divergentorientation, such that the step of non-covalently linking the promoterand the terminator to the open reading frame results in a circularexpression element.
 88. A linear or circular expression element producedby a method comprising: a) obtaining a DNA segment comprising an openreading frame; and b) linking the open reading frame to a promoter and aterminator to create a linear or circular expression element.
 89. Theexpression element of claim 88, wherein the DNA segment is obtained froma process involving PCR®.
 90. The expression element of claim 89,wherein the PCR® reaction is primed with oligonucleotides having acomplementary stretch incorporating deoxyuridines.
 91. The expressionelement of claim 90, wherein the deoxyuridines are incorporated everythird position of the complementary stretch.
 92. The expression elementof claim 88, wherein the open reading frame is non-covalently linked tothe promoter and the terminator.
 93. The expression element of claim 92,wherein the non-covalent linkage is performed by: a) obtaining a PCR®product comprising the open reading frame, which PCR® product isobtained by amplification from at least one primer that hascomplementary stretches comprising deoxyuridines with uracil-DNAglycosylase to create overhangs to which the promoter and terminator canlink; b) providing a promoter and a terminator; c) non-covalentlylinking the promoter and the terminator to the open reading frame tocreate the linear or circular expression element.
 94. The expressionelement of claim 93, wherein the deoxyuridines are incorporated at everythird position of the complementary stretches.
 95. The expressionelement of claim 93, wherein the primer that has complementary stretchescomprising deoxyuridines comprises the promoter and the terminator indivergent orientation, such that the step of non-covalently linking thepromoter and the terminator to the open reading frame results in acircular expression element.
 96. A linear or circular expression elementcomprising a DNA segment comprising an open reading frame and a promoterand terminator non-covalently linked to said open reading frame.